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(Received for publication, November 20,
1995; and in revised form, February 23, 1996) From the
Dystrophin serves a variety of roles at the cell membrane
through its associations, and defects in the dystrophin gene can give
rise to muscular dystrophy and genetic cardiomyopathy. We investigated
localization of cardiac dystrophin to determine potential intracellular
sites of association. Subcellular fractionation revealed that while the
majority of dystrophin was associated with the sarcolemma, about 35% of
the 427-kDa form of dystrophin was present in the myofibrils. The
dystrophin homolog utrophin was detectable only in the sarcolemmal
membrane and was absent from the myofibrils as were other sarcolemmal
glycoproteins such as adhalin and the sodium-calcium exchanger.
Extraction of myofibrils with KCl and detergents could not solubilize
dystrophin. Dystrophin could only be dissociated from the myofibrillar
protein complex in 5 M urea followed by sucrose density
gradient centrifugation where it co-fractionated with one of two
distinctly sedimenting peaks of actin. Immunoelectron microscopy of
intracellular regions of cardiac muscle revealed a selective labeling
of Z-discs by dystrophin antibodies. In the genetically determined
cardiomyopathic hamster, strain CHF 147, the time course of development
of cardiac insufficiency correlated with an overall 75% loss of
myofibrillar dystrophin. These findings collectively show that a
significant pool of the 427-kDa form of cardiac dystrophin was
specifically associated with the contractile apparatus at the Z-discs,
and its loss correlated with progression to cardiac insufficiency in
genetic cardiomyopathy. The loss of distinct cellular pools of
dystrophin may contribute to the tissue-specific pathophysiology in
muscular dystrophy.
The dystrophin gene encodes a 427-kDa protein in muscle and
brain tissues. Absence of this full-length form of dystrophin causes
severe, progressive muscle weakness in patients with Duchenne's
or Becker's muscular
dystrophies(1, 2, 3) . These patients may
also experience dilated cardiomyopathy, which in some cases may be much
more severe than the myopathy(37, 38, 39) .
The precise physiological role of dystrophin is not fully known
although, due to its subsarcolemmal localization, it has been proposed
to serve a variety of functions such as providing stability to the
membrane and signal
transduction(4, 9, 10, 14) .
Dystrophin forms a tight association with a glycoprotein complex
embedded in the plasma membrane(5, 6, 7) .
One of these glycoproteins, In addition to its role in membrane
stability and receptor clustering, recent studies imply that dystrophin
serves a unique role in signal transduction by localizing nitric-oxide
synthase to the sarcolemma (15) . In order to elucidate the
cellular roles of dystrophin, it is paramount to precisely define its
associations. Although it is well documented that dystrophin localizes
at the plasma membrane in a variety of
cells(16, 17, 18, 19) , studies
indicate that there may be tissue- and cell-specific differences in
dystrophin localization. For example, recent studies using confocal
imaging (20, 21, 22, 23) and
immunogold labeling (23) have shown that in addition to the
sarcolemma, a substantial pool of dystrophin also localizes at the
transverse tubules in cardiac muscle. Furthermore, cardiac dystrophin
was found to be distributed in three distinct subcellular pools, a
cytoplasmic pool, a membrane-bound pool not associated with
WGA(
Subcellular fractions from fresh rabbit hearts were isolated as
described under ``Experimental Procedures.'' SDS-PAGE
analysis of the protein composition of the different fractions obtained
during myofibril isolation is shown in Fig. 1A. The
cardiac homogenate (lane H) was centrifuged to obtain a
supernatant fraction (lane S1) and a pellet fraction (lane
P1). The pellet was washed extensively with buffer II to obtain
supernatant (lane S2) and pellet (lane P2). Fraction
P2 was further extracted with Triton X-100 to obtain supernatant (lane S3) and pellet (lane P3). The final product
represented isolated myofibrils (MF). The MF fraction
consisted predominantly of myosin (200 kDa) and actin (42 kDa). Other
cytoskeletal proteins, such as
Figure 1:
Presence of dystrophin in cardiac
myofibrils. A, Coomassie Blue staining of SDS gels
representing cardiac fractions obtained during isolation of myofibrils.
Each lane was loaded with 60 µg of proteins, and the proteins were
separated on a 6.25% SDS-polyacrylamide gel. H, homogenate; S1, supernatant fraction of the homogenate (buffer I wash); P1, pellet of the homogenate; S2, supernatant of the
buffer II wash; P2, pellet of the buffer II wash; S3,
supernatant of the Triton X-100 (buffer III) wash; P3, pellet
of the Triton X-100 wash; MF, the isolated myofibrils washed
with the suspension buffer. Standard protein markers (bands from the top to the bottom represent 211, 117, 81, 49, and 31
kDa, respectively). B, immunoblots of cardiac fractions
obtained from A. Panel a, anti-
The protein composition of
the myofibril preparation was further examined by immunoblotting with
various antibodies directed against known myofibrillar proteins and
dystrophin. Gels with identical sample loading to that shown in Fig. 1A were electroblotted onto nitrocellulose and
immunostained with the various antibodies (Fig. 1B).
Anti-desmin and anti- To identify any cross-contamination of the myofibril
fractions with the sarcolemmal membrane, we investigated the presence
of the Na
Figure 2:
Presence of dystrophin in cardiac
sarcolemmal and myofibrillar fractions. A, Coomassie Blue
staining of the myofibrillar and sarcolemmal fractions obtained from
the same rabbit heart. Each lane was loaded with 5 µg of proteins,
and samples were separated on a 6.25% SDS-polyacrylamide gel. H, homogenate; S1, supernatant fraction of the
homogenate (buffer I) wash; P1, pellet of the homogenate; MF, the isolated myofibrils; C, crude cardiac
sarcolemma; SL, purified cardiac sarcolemma. Standard protein
markers (bands from the top to the bottom represent
211, 117, 81, 49, and 31 kDa, respectively). B, immunoblots of
cardiac myofibrillar and sarcolemmal fractions. Each lane was loaded
with 60 µg of protein, and samples were separated on a 6.25%
SDS-polyacrylamide gel and transferred to nitrocellulose for Western
blotting. Panel a, anti-
As observed
with the previous fractionation procedure, desmin and Immunoblotting of the various fractions
obtained using the modified protocols revealed the presence of the
427-kDa form of dystrophin in the purified sarcolemma (Fig. 2B, lane SL) and in the myofibrillar
fraction (Fig. 2B, lane MF). Although the
intensity of dystrophin staining was slightly lower in the myofibrillar
fraction than in the purified sarcolemma, we reasoned that
quantitatively it may represent a substantial fraction of the total
dystrophin content of cardiac muscle. Western blots of the fractions
shown in Fig. 2B were therefore digitally scanned to
quantitate the signal intensity of the immunostaining per lane, and
this was normalized to the microgram of protein loaded per lane. These
values were then multiplied by the total amount of protein present in
each fraction to determine the relative amount of dystrophin,
Na Dystrophin is
known to localize at the sarcolemma in association with membrane
glycoproteins. We examined the glycoprotein composition of the MF and
SL fractions in a WGA overlay reaction (Fig. 3a). WGA
staining of glycoproteins of various molecular mass was detected in the
homogenate (lane H) and crude membranes (lane C), and
this intensified in the purified sarcolemmal membranes (lane
SL). There was weak WGA staining detected in the pellet (lane
P1) of the homogenate of glycoproteins of 160, 220, and 300 kDa,
and these were seen to enrich in the myofibrils (lane MF)
purified from this pellet. These glycoproteins did not appear in the
soluble fraction (lane S) or the crude (lane C) and
purified membranes (lane SL). Immunoblotting with an antibody
against adhalin, a 50-kDa dystrophin-associated sarcolemmal
glycoprotein (Fig. 3b), revealed that while this
glycoprotein was detectable in the homogenate (lane H) and
enriched in the sarcolemmal membrane (lane SL), it was absent
from the myofibrils (lane MF).
Figure 3:
Distribution of glycoproteins in cardiac
fractions. Panel a, WGA overlay and panel b, Western
blotting with adhalin (Ad) antibodies of cardiac myofibrils (MF), sarcolemma (SL), crude membranes (C),
and supernatant (S1) and pellet (P1) isolated from
the same heart homogenate (H). Arrowheads indicate
glycoproteins of 160, 220, and 300 kDa enriched in MF. The data are
typical of two independent experiments.
Figure 4:
Effect of salt extraction on myofibrillar
dystrophin. Protein patterns (panel a) and immunoblots (panel b) of the fractions obtained from the isolation of
myofibrils with or without a KCl wash. Isolated myofibrils (MF) without KCl extraction; supernatant (S) and
pellet (P) obtained after KCl extraction of MF. Dys,
dystrophin. Dystrophin antibody was applied at a dilution of 1:500. The
data are typical of three independent experiments. My, myosin;
The dissociation of dystrophin
from myofibrils was further examined by solubilizing the myofibrillar
fraction in 5 M urea, followed by separating the proteins in a
5-30% continuous sucrose density gradient. Protein assay
indicated that more than 85% of total myofibrillar protein remained
soluble in the urea buffer after centrifugation at 100,000
Figure 5:
Sedimentation of myofibrillar dystrophin
in a sucrose density gradient. Isolated myofibrils were solubilized in
urea and separated in a 5-30% sucrose density gradient. Numbers
at the bottom of the figure indicate the fractions collected
from the bottom (lane 1) to the top (lane 23) of the
sucrose gradient. Coomassie Blue staining (a) and
immunoblotting (b) of proteins in the various fractions was
examined. Dystrophin antibody was applied at a dilution of 1:500. My, myosin;
Figure 6:
Dystrophin in skeletal and cardiac muscle.
Coomassie Blue staining of SDS gels (a) and immunoblotting (b) of cardiac (c) and skeletal (s) muscle
homogenate (H), myofibrils (MF), and sarcolemma (SL). Dystrophin antibody was applied at a dilution of 1:500. Dys, dystrophin; My, myosin;
Figure 7:
Distribution of utrophin and dystrophin in
the heart. All fractions were from the same rabbit heart. Each lane was
loaded with 60 µg of proteins, and samples were separated on a
6.25% SDS-polyacrylamide gel. H, homogenate; S3,
supernatant fraction of the Triton X-100 wash; MF, isolated
myofibrils; C, crude cardiac sarcolemma; SL, purified
cardiac sarcolemma. Dystrophin (Dys) antibody was applied at a
dilution of 1:500. Monoclonal antibody against utrophin (Utr)
was applied at a dilution of 1:500. The data are typical of three
independent experiments.
Figure 8:
Localization of dystrophin at the
sarcomere monitored with immunogold 5-µm labeling on ultrathin
cryosections. A typical of 50 micrographs generated from four different
hearts is shown. Gold label (black dots) is seen along the Z
line with some labeling present at the sarcolemma. 58,750
Figure 9:
Immunoblot analysis of dystrophin
expression in normal and cardiomyopathic hamster heart. Subcellular
fractions were isolated from cardiomyopathic CHF 147 strain (M) and normal hamster hearts (C) and separated in
SDS-PAGE and analyzed in Western blots with affinity purified
dystrophin antibodies. Lane S, sarcolemma; lane H,
heart homogenate; lanes MF, myofibrils. Identical amounts of
protein (40 µg) per lane were loaded between the myopathic and
control groups. The results shown are typical of two to three
independent experiments from groups of animals at day
180.
Figure 10:
Evidence for myofibrillar dystrophin loss
in the development of cardiomyopathy. A, myofibrils were
prepared from hearts of cardiomyopathic hamsters (M) and
normal control (C) hearts at ages 30, 60, 120, and 180 days
and analyzed in Western blots with affinity purified dystrophin
antibodies. B, Western blots were quantified by densitometry,
and dystrophin content was plotted as a function of age in the normal (C dys) and cardiomyopathic (M dys) hamster. The
desmin content in normal (C des) and cardiomyopathic (M
des) hamsters on the same blot was also quantified by Western
blotting and densitometry at the various ages shown. The results are
typical of two to three independent experiments performed on two to
three different groups of animals.
The present results demonstrate that a distinct pool of
dystrophin molecules associated with the myofibrillar fraction and
localized to the Z-discs in cardiac muscle. Whereas a majority of the
dystrophin is localized at the surface membrane in association with
glycoproteins, our results show that dystrophin at the Z-discs exists
in the absence of any of these glycoproteins. Myofibrillar localization
of dystrophin was exclusive to cardiac muscle, and its loss correlated
with the development of cardiac insufficiency in the genetically
determined cardiomyopathic hamster. Dystrophin was tightly
associated with the cardiac myofibrillar fraction that was enriched in
cytoskeletal proteins such as The tight association
of cardiac dystrophin with the myofibrillar fraction suggests that
dystrophin may play a unique role in the structure or function of the
cardiac myofibril and that this pool of dystrophin may associate with
proteins that are distinct from those of the sarcolemmal membrane
dystroglycan complex. Accordingly, we found that adhalin, the 50-kDa
dystrophin-associated glycoprotein, was present only in the sarcolemmal
and not the myofibrillar fractions. The pool of myofibrillar dystrophin
cannot therefore be associated with any of the glycoproteins found in
the dystroglycan complex as is the case for a sarcolemmal pool of
dystrophin. The model for the structure of the dystrophin-containing
membrane cytoskeletal complex suggests that dystrophin is anchored in
this complex via actin at its N-terminal domain and to one or more
components of the membrane glycoprotein complex via its C-terminal
domain. It will therefore be of interest to determine whether actin
alone anchors dystrophin so tightly to the cardiac myofibrils or
whether an additional dystrophin-binding protein is present in the
Z-discs. In this regard dystrophin has been shown to localize
nitric-oxide synthase to the sarcolemmal membrane and proposed to serve
a role in nitric oxide signaling(15) . Whether dystrophin at
the Z-disc can anchor such signaling molecules at the sarcomere is now
under investigation. The origin and the nature of glycoproteins of 160,
220, and 300 kDa in the myofibrillar fraction are intriguing. These
glycoproteins could not be extracted with detergents and high salt.
Whether dystrophin can associate with any of these glycoproteins to
anchor them at the Z-disc remains to be investigated. The
dystrophin-related protein, utrophin, was exclusively distributed in
the sarcolemmal membrane fraction of cardiac muscle and was absent from
the myofibrillar fraction. This is consistent with the
immunohistochemical localization of utrophin in heart
sarcolemma(47) . The fact that utrophin was absent from the
cardiac myofibrillar fraction argues against artificial redistribution
of dystrophin during the subcellular fractionation procedure. The
expression of substantial amounts of utrophin in normal adult cardiac
muscle is another noteworthy difference between cardiac and skeletal
muscle(29) . Our finding that about 35% of the total cardiac
muscle content of the 427-kDa form of dystrophin is associated with
myofibrils points to a unique role for dystrophin in the
structure/function of the cardiac myofibril. It is evident that
dystrophin is a component not only of the membrane cytoskeleton but
also of the intracellular cytoskeletal network that organizes
myofibrils at the level of the Z-disc. It is of interest that the most
frequent cardiac abnormality observed in dystrophin deficiency is
dilated cardiomyopathy(37, 38, 39) , a
condition associated with cytoskeletal disorganization as well as
irregular sarcomeric and Z-disc structures(48) . The
cardiomyopathic hamster that exhibits autosomal recessive
cardiomyopathy and experiences muscular dystrophy has been widely used
as a model system. While the precise genetic defects remain to be
defined, several biochemical abnormalities associated with
Ca
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12364-12371
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-dystroglycan, has been shown to bind
to the G-domains of laminin (8) and agrin to mediate
clustering of acetylcholine receptors(9, 10) . The
association of a pool of subsarcolemmal dystrophin with laminin in the
extracellular matrix via the membrane glycoproteins may be vital for
linking the cytoskeleton to the extracellular matrix, and the
disruption of this complex leads to increased membrane
fragility(4, 9, 10) . Alterations in
components of the dystrophin-glycoprotein-laminin complex, such as the
laminin- 2 gene product, and a 50-kDa dystrophin-associated
glycoprotein, adhalin, are associated with muscle membrane instability
and muscular dystrophy(11, 12, 13) . A
deficiency in adhalin and disruption of the membrane
dystrophin-glycoprotein complex has also been reported in the
cardiomyopathic hamster that experiences both hereditary cardiomyopathy
and myopathy(14) .
)-binding glycoproteins, and a membrane-bound pool
associated with WGA-binding glycoproteins(20, 22) . In
view of the notion that dystrophin may serve diverse roles through its
associations, here we demonstrate a selective pool of dystrophin
molecules that associate with the myofibrils and localize to the
Z-discs in cardiac muscle. Furthermore, the specific loss of
myofibrillar dystrophin during progression to cardiac insufficiency in
the cardiomyopathic hamster CHF 147 suggests that dystrophin may serve
a unique role at the level of the sarcomere in addition to its recently
described membrane functions. The distinct pools of dystrophin and
their selective loss may be of significance in explaining the
tissue-specific pathophysiological responses in muscular
dystrophy(24) .
General Conditions
All muscle preparations were
performed at 4 °C, and all buffers contained the following protease
inhibitors, 1 mM benzamidine, 1 mM iodoacetamide, 1
µg/ml trypsin inhibitor, 2 µg/ml leupeptin, 1 µg/ml
pepstatin A, and 0.2 mM phenylmethanesulfonyl fluoride.Isolation of Myofibrils from Cardiac and Skeletal
Muscle
Myofibrils were prepared as described(25) .
Ventricular muscle of rabbit heart or fast-twitch skeletal muscle
(4-5 g) from rabbits was homogenized in 20 volumes of buffer I
(39 mM sodium borate, 25 mM KCl, 5 mM EGTA,
and 1 mM dithiothreitol (DTT), pH 7.1) with the Brinkman
polytron (PT-20) for 15 s, twice. The homogenate was centrifuged at
1,500 
g for 12 min and the pellet was re-extracted.
The pellet was resuspended in 20 volumes of buffer II (39 mM sodium borate, 25 mM KCl, and 1 mM DTT, pH 7.1)
and centrifuged at 1,500 g for 12 min. This step was
repeated and the pellet further extracted for 30 min with Triton X-100
buffer (39 mM sodium borate, 25 mM KCl, 1 mM DTT, and 1% Triton X-100, pH 7.1). The material was filtered and
centrifuged at 1,500 g for 12 min and the pellet
re-extracted once more. The pellet was then washed twice with
suspension buffer (10 mM Tris, 100 mM KCl, and 1
mM DTT, pH 7.1). The final pellet (myofibrils) was resuspended
in low-salt buffer (10 mM Tris, 100 mM KCl, 1 mM DTT, and 20% glycerol, pH 7.1) at 2-3 mg/ml and stored at
-78 °C for further analysis.Purification of Sarcolemmal Membrane
Cardiac
sarcolemmal membranes from rabbit hearts were prepared by differential
and discontinuous sucrose density gradient as described
previously(26) . The isolation of crude sarcolemmal membranes
from rabbit skeletal muscle was as described by Seiler and
Fleischer(27) .Separation of Myofibrillar Proteins
Approximately
2.5 mg of isolated myofibrils were solubilized in 4 ml of urea buffer
(0.5 M KCl, 50 mM Tris, 5 M urea, 1 mM EGTA, 10 mM ATP, 1 mM DTT, and protease
inhibitors, pH 8.0) and centrifuged at 100,000 g for 1
h at 4 °C. The supernatant was loaded on a 5-30% continuous
sucrose density gradient and centrifuged at 200,000 g for 24 h at 4 °C. The sucrose gradient fractions were
collected and concentrated using Centricon-30 tubes (Amicon) and
protein composition analyzed by 7.5% SDS-PAGE.Cardiomyopathic Animals
Cardiomyopathic hamsters
of the CHF 147 line (derived from the UM-X 7.1 line) were obtained from
Canadian Hybrid Farms (Nova Scotia, Canada). Hearts from
cardiomyopathic and normal hamsters at various ages (30, 60, 120, and
180 days) were obtained and the homogenate, membrane, and myofibrillar
fractions isolated as described above.Antibodies and Western Blotting
Polyclonal
antibody against dystrophin was raised in rabbits by injecting
synthesized antigenic peptide corresponding to the C-terminal amino
acid sequence (SSRGRNTPGKPMREDTM) of the human dystrophin, conjugated
to keyhole limpet hemocyanin as described(22) . Monoclonal
antibody Mancho-7 against utrophin was a gift from Dr. Glen Morris and
has been previously characterized(28, 29) . The
monoclonal antibody against cardiac Na
-Ca
exchanger was a gift from Dr. K. D. Philipson, University of
California Los Angeles. The monoclonal antibody against -actinin
and alkaline phosphatase conjugated anti-mouse IgG antibody were
purchased from Sigma. The monoclonal antibody against desmin was a gift
from Dr. D. Parry, University of Ottawa, and anti-adhalin was provided
by Dr. S. Carbonetto, McGill University. Protein samples were separated
by SDS-PAGE in the presence of dithiothreitol and transferred to
nitrocellulose. The nitrocellulose was blocked with 5% Blotto in
Tris-buffered saline and incubated with primary antibody for 1 h. The
membranes were washed three times with Tris-buffered saline-Tween
(0.05%) solution and incubated with alkaline phosphatase-conjugated
second antibody for 1 h. Color was developed with the mixture of nitro
blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. For WGA
overlay nitrocellulose was blocked with 5% bovine serum albumin for 1 h
and then incubated with horseradish peroxidase-conjugated WGA for 1 h.
Color was developed with a mixture of 4-chloro-1-naphthol and hydrogen
peroxide.Immunolabeling at the Electron Microscopic
Level
Adult rabbit hearts were rapidly excised and placed in
oxygenated Tyrode's solution as described
previously(23) . Fresh solution was added containing 2%
formaldehyde, 0.01% glutaraldehyde for 20 min. Papillary muscles were
removed, minced into small pieces, and fixed for an additional 40 min.
Samples of papillary muscles were immersed in 2.3 M sucrose
for 4 h and then rapidly frozen by immersion in liquid propane as
described previously(30) . The sections (70-100 µm)
were mounted on Formvar-coated grids, incubated with blocking solution,
followed by incubation with antibody at room temperature. After
washing, the sections were incubated in goat-anti-rabbit IgG-5
m
gold conjugate (Amersham Corp.). The sections were post-fixed in 2%
glutaraldehyde in phosphate-buffered saline, rinsed, counterstained,
coated with a thin layer of 1.5% methylcellulose, and allowed to
air-dry. In order to quantitate the number of gold particles over the
Z-disc versus gold distributed in other areas of the
myofibrils, we examined 50 micrographs from 4 papillary muscles. In
total, we examined 63 µm of Z-discs and 63 µm of A band region
of the contractile proteins. All electron microscopic observations were
with a JEOL 100CX.
Subcellular Fractionation Reveals Dystrophin Is
Associated with the Myofibrillar Fraction
We raised antibodies
to the C-terminal 17 amino acids of the skeletal dystrophin sequence.
This sequence is exclusive to the 427-kDa form of dystrophin and is not
present in any other protein including -actinin(31) . The
antibodies were affinity purified on the peptide and specifically
recognized dystrophin from skeletal muscle, heart, and brain as we
described previously(20, 22, 23) . We used
the purified antibodies to investigate the localization of dystrophin. -actinin (105 kDa) and desmin (55
kDa) were also present in this fraction.
-actinin antibody
(1:20,000) and anti-desmin ab (1:1,000); Panel b,
anti-Na-Ca exchanger antibody
(1:1000); Panel c, anti-dystrophin antibody (1:1000). The
mobility of myosin (My), -actinin (-A),
actin (Ac), desmin (Des),
Na-Ca exchanger (Na-Ca) and
dystrophin (Dys) is indicated.
-actinin staining indicated that these
proteins co-fractionated in a similar manner. The pellets of the washes
and the isolated myofibrils stained strongly for desmin and
-actinin (Fig. 1B, panel a, lanes P1, P2, P3, and MF), whereas the supernatant fractions had no
detectable desmin signal and stained only faintly for -actinin (Fig. 1B, lanes S1, S2, and S3). Staining with
anti-dystrophin antibodies was readily detectable in the cardiac
homogenate, and it became stronger in the myofibrillar pellets obtained
after extraction with buffer I and buffer II (Fig. 1B, panel
c, lanes P1 and P2). In contrast to this, there was
little dystrophin staining found in the corresponding supernatant
fractions (Fig. 1B, lanes S1 and S2).
Furthermore, the nonionic detergent, Triton X-100, failed to extract a
significant amount of dystrophin from the pellet fraction (Fig. 1B, lane S3). After the Triton X-100
extractions the isolated myofibrils still contained a significant
amount of dystrophin staining (Fig. 1B, lane
MF).-Ca exchanger which is a
transmembrane glycoprotein. Immunostaining with cardiac
Na-Ca exchanger antibody revealed
that some of this plasma membrane marker remained in the myofibril
pellet during the first two washes of the isolation procedure (Fig. 1B, panel b, lanes P1 and P2).
However, this residual membrane glycoprotein was completely removed
from the pellet fraction by extraction with Triton X-100 (Fig. 1B, lanes S3 and P3). This
immunostaining pattern for the various polypeptides was consistent in
all 18 different myofibril preparations examined.Proportion of Total Cardiac Dystrophin Associated with
the Myofibrillar Fraction
To determine the relative amount of
dystrophin present in the sarcolemmal membrane and myofibrillar
fractions, the myofibril isolation protocols were modified to prepare
both of these fractions from the same rabbit heart. A Coomassie
Blue-stained SDS gel containing these fractions is shown in (Fig. 2A). The cardiac muscle was first used to purify
a crude sarcolemmal fraction, which was then subjected to discontinuous
sucrose density gradient centrifugation to obtain a purified
sarcolemmal fraction. The pellet obtained from the cardiac homogenate (Fig. 2A, lane P1) was used to isolate
myofibrils (lane MF), whereas the supernatant was used to
prepare crude sarcolemmal membranes (lane C) which were then
subjected to discontinuous sucrose gradient to purify the sarcolemma (lane SL). The myofibril fraction contained mainly myosin,
-actinin, and actin (Fig. 2A, lane MF)
similar to the protein composition of the myofibrillar fraction shown
in Fig. 1. The crude sarcolemmal fraction contained
substantially less of these myofibrillar proteins (Fig. 2A, lane C), and they were further
diminished in the purified sarcolemmal fraction as judged by protein
staining (Fig. 2A, lane SL).
-actinin antibody (1:5000); Panel b, anti-desmin antibody (1:1000); Panel c,
anti-Na-Ca exchanger antibody
(1:1,000); anti-dystrophin antibody (1:1000). Dys, dystrophin; NaCa, Na-Ca exchanger; Des, desmin; -A, -actinin; My,
myosin. The data are typical of four independent
experiments.
-actinin
staining was intense in the myofibrillar fraction (Fig. 2B, lane MF) when compared with the
homogenate or sarcolemmal fractions. Immunostaining for the
Na-Ca exchanger revealed a 120-kDa
band in the crude sarcolemmal fraction (Fig. 2B, lane C) that was significantly stronger in the purified
sarcolemmal fraction (Fig. 2B, lane SL). A
70-kDa band also present with the 120-kDa band in the SL fraction was
most likely a degradation product of the
Na-Ca exchanger(32) .
Na-Ca exchanger staining was
undetectable in the myofibrillar fraction (Fig. 2B, lane MF). These findings indicate that the modified protocol
worked well and gave a clear separation of the sarcolemmal and
myofibrillar fractions.-Ca exchanger, desmin, and
-actinin present in the homogenate and the various fractions
prepared from it. The percent of dystrophin present in the membrane and
myofibrillar fractions was compared with the various markers. The
results show that approximately 35% ± 5 of the total dystrophin
present in the homogenate was recovered in association with the
myofibrillar fraction, whereas the recovery of desmin and -actinin
in this fraction was calculated to be 88% ± 17 and 80% ±
18, respectively (n = 4). The total amount of
homogenate dystrophin that was recovered in the sarcolemmal fraction
was approximately 55%, compared with a 90% recovery of the
Na -Ca exchanger.
Dystrophin Is Tightly Associated with the Myofibrillar
Fraction
To determine how tightly dystrophin was associated with
proteins of the myofibrillar fraction, we examined the conditions
required to dissociate dystrophin from other components of this
fraction. We first examined the effect of high-salt extraction. The
isolated myofibrils were extracted with 0.7 M KCl for 60 min
at 4 °C, and the soluble proteins were separated from the insoluble
material by centrifugation. The KCl wash solubilized approximately 30%
of the myofibrillar proteins, and the protein composition of the
soluble versus the insoluble fractions was examined in SDS
gels and Western blots (Fig. 4). The polypeptide patterns of the
myofibrils, supernatant, and pellet were quite similar as seen by
Coomassie Blue staining (Fig. 4a, lanes MF, S, and P). However, immunoblotting with dystrophin antibodies
detected the polypeptide only in the myofibrils and the insoluble
pellet and not the soluble material (Fig. 4b). Similar
results were seen when detergents, such as Triton X-100, Nonidet P-40,
deoxycholate, and digitonin, were used to extract the myofibrils
together with KCl (data not shown).
-A, -actinin; Ac,
actin.
g for 1 h. Western blot analysis confirmed that the dystrophin
was in the soluble fraction (data not shown). The protein patterns of
the various fractions obtained from the sucrose gradient fractionation
are shown in Fig. 5a. Myosin, -actinin, and actin
were the major components in the sucrose gradient fractions. A single
myosin peak was clearly separated from a single -actinin peak.
Actin appeared in two distinct peaks in the gradient. The first actin
peak (Fig. 5a, Fractions 14-19) was located near
the top of the sucrose gradient. This represented most of the actin
present in the myofibrillar fraction. A second smaller peak was
localized near the bottom of the sucrose gradient (Fig. 5a, Fractions 6-8). This peak did not
precisely correspond to the myosin peak, although it substantially
overlapped it. The lower actin peak did, however, precisely correspond
to a single dystrophin peak in the gradient (Fig. 5, a and b, Fractions 6-8).
-A, -actinin; Ac,
actin; Dys, dystrophin. The data are a typical of four
independent experiments.
Comparison with Skeletal Muscle
To determine
whether some cofractionation of dystrophin with the myofibrillar
fraction may also occur in skeletal muscle, various subcellular
fractions were prepared from rabbit fast twitch skeletal muscle. The
protein patterns of the skeletal muscle fractions are shown in Fig. 6a. Skeletal myofibrils (lane
MF
) contained mostly myosin, -actinin, and actin,
similar to cardiac myofibrils (lane MF
). In the
skeletal muscle sarcolemma (SL) and cardiac
sarcolemma (SL) there was much less myosin, actin,
and -actinin compared with the myofibrils. Immunoblotting with
anti-dystrophin antibodies indicated dystrophin staining was
significantly enriched in skeletal muscle sarcolemma, but in contrast
to cardiac myofibrils (Fig. 6b, lane
MF) no dystrophin was detected in the myofibrils from
skeletal muscle (Fig. 6b, lane
MF). Dystrophin staining was evident in the
homogenates from both types of muscle (lanes H and H).
-A,
-actinin; Ac, actin. The data are typical of three
independent experiments.
Utrophin Is Present Exclusively in the Sarcolemmal
Fraction of Cardiac Muscle
The distribution of the dystrophin
homolog utrophin in cardiac fractions was compared with that of
dystrophin. Western blot analyses with utrophin antibodies revealed
that utrophin staining was present exclusively in the purified
sarcolemmal fraction (Fig. 7, lane SL) and was notably
absent from the cardiac myofibril fraction (Fig. 7, lane
MF), whereas dystrophin staining was readily detectable in the
myofibrils, sarcolemma, homogenate (H), and crude membranes (C). It should be noted that overdevelopment of the reaction
resulted in minor labeling of utrophin in the homogenate and crude
membranes, but no utrophin was detected in myofibrils.
Immunogold Labeling Shows Dystrophin Localizes to
Z-discs
To determine whether dystrophin was localized
preferentially to one region within myofibrils, we examined the
localization of dystrophin in intracellular regions of cardiac muscle
using immunogold-labeled antibodies directed against dystrophin. Fig. 8is a typical electron micrograph that illustrates the
distribution of gold over the contractile proteins. Immunogold labeling
was clearly more abundant over the Z-disc than in any other region of
the myofibrils. In 50 micrographs (4 hearts, 4 sections per heart for a
total of 63 µm counted), we found a total of 373 gold particles
over the Z-discs and 104 particles over other areas of the contractile
proteins. We have previously documented the localization of dystrophin
at the sarcolemma and transverse tubules using electron
microscopy(23) .
.
Myofibrillar Dystrophin and Cardiac Derangement in
Genetic Cardiomyopathy
In order to determine a potential role
for myofibrillar dystrophin in cardiac derangement, we examined the
expression of dystrophin in the genetically determined cardiomyopathic
hamster which experiences severe cardiac insufficiency. The
time-dependent developments in cardiac derangements in the CHF 147 line
(descendents of the UM-X7 line) of cardiomyopathic hamsters has been
well documented(33, 34, 35, 36) .
The CHF 147 strain of hamsters is particularly relevant here because
these animals experience dilated cardiomyopathy as well as muscular
dystrophy symptoms similar to that seen in some human cases of muscular
dystrophy(37, 38, 39) . This strain of
hamsters exhibits the following chronology of events in the myocardium,
cardiac myolysis at 60 days followed by cardiac dilation at
80-120 days and cardiac insufficiency at 150 days and in older
animals. Previous studies have documented a number of ultrastructural
alterations at the cardiac insufficiency stage including
disorganization of the myofibrillar arrangement where the Z-discs were
noted to be irregular(33, 34, 35) . We
examined the expression of dystrophin which may account for some of
these changes in the cardiomyopathic hamster heart. Western blot
analysis (Fig. 9) shows that at day 180 there was a marked
decrease in the dystrophin staining in the homogenate (lane H)
from cardiomyopathic hamsters (M) compared with their
age-matched control (C). The membranes (lane S)
isolated from the homogenates of these animals also indicated a
difference in dystrophin staining between the myopathic and normal
animals, although the decrease in staining intensity was not as marked
as that noted in the homogenates. Dystrophin staining in the
myofibrillar fraction (MF) indicated that there was a marked
decrease in the myofibrillar dystrophin in the cardiomyopathic hamster
heart. Quantification of the data using densitometry indicated that the
total dystrophin content in the cardiomyopathic hamster heart was
reduced by 63% (average n = 2) which was reflected as a
drop in membrane dystrophin of 28% (average n = 2)
while myofibrillar dystrophin decreased by 75% (average n = 2). We further examined the time course of dystrophin
loss from the myofibrillar fraction as these animals develop cardiac
insufficiency. Fig. 10A shows dystrophin staining in
myofibrils isolated from cardiomyopathic hamsters (M) and
age-matched controls (C) at day 30, 60, 120, and 180. The data
were quantified (Fig. 10B), and it can be seen that at
day 30 the dystrophin content in the myofibrils from cardiomyopathic
and normal hearts was similar, and thereafter there was a progressive
time-dependent decrease in dystrophin with a 75% ± 7 (n = 3) drop at day 180. The expression of desmin was noted to
be similar in the two groups of animals (Fig. 10B). The
time course of myofibrillar dystrophin loss closely paralleled the
decrease in contractile function reported for the CHF 147 hamster heart
muscle (36) while the time-dependent accumulation of
myofibrillar dystrophin in the normal hamster (Fig. 10)
correlated with the age-dependent increase in contractile performance
documented previously(36) .
-actinin and desmin. While
dystrophin associated with the sarcolemmal membrane was soluble in
detergents such as digitonin, Nonidet P-40, Triton X-100, and
deoxycholate as previously
reported(22, 40, 41, 42) , these
treatments failed to extract dystrophin from cardiac myofibrils. High
concentrations of KCl solubilized approximately 30% of the protein
content of isolated cardiac myofibrils, but dystrophin was not present
in the soluble fraction. Dissociation of dystrophin from other proteins
in the myofibrillar fraction could only be achieved by solubilization
of myofibrils in buffer containing 5 M urea followed by
separation of dissolved proteins on a sucrose density gradient. On the
sucrose gradient, the peak of myofibrillar dystrophin followed a
pattern different from the myosin and -actinin peaks, but it
coincided with one of two distinct actin peaks, suggesting that
myofibrillar dystrophin may be associated with actin. Dystrophin is
known to bind actin via its N-terminal
domain(43, 44, 45) . Various isoforms of
actin are expressed by mature striated muscle. In addition to
muscle-specific actin present in the thin filaments, actin is also
present in the Z-disc and in the cytoskeletal scaffold surrounding
myofibrillar bundles(46) . Further work is therefore required
to determine the significance of the existence of two distinct pools of
actin in the myofibrillar fraction and the selective association of
dystrophin with one of these pools. Given our present observation of
immunogold labeling of Z-discs with dystrophin antibody in cardiac
muscle, the pool of actin cofractionating with dystrophin on the
sucrose gradient may represent Z-disc actin. overload have been noted(49) . The CHF 147
strain of hamsters experience dilated cardiomyopathy and have been
shown to exhibit age-dependent morphological alterations in the
sarcomeric and Z-disc arrangements that correlate with the decrease in
contractile function and progression to cardiac insufficiency (33, 34, 35, 36) . Our results
indicating the loss of myofibrillar dystrophin during the time course
of these cardiac derangements suggest that dystrophin at the Z-disc may
play an essential role in maintaining structural integrity, and its
loss may contribute to the more severe cardiomyopathy compared with the
milder muscular dystrophy experienced by these animals. Furthermore,
the time-dependent increase in myofibrillar dystrophin paralleled the
enhanced contractile performance documented for the normal hamster
heart(36) , implying that dystrophin serves an important role
in the contractile apparatus. It should be noted that the expression of
desmin was essentially unaltered during the progression of cardiac
insufficiency in the CHF 147 hamster heart, and this is consistent with
results obtained in human-dilated cardiomyopathy(48) . The CHF
147 hamster is related to the BIO 14.6 cardiomyopathic strain that has
been reported to completely lack adhalin expression in both cardiac and
skeletal muscle with either no change or only a slight decrease in
membrane dystrophin levels(14, 50) . The lack of
adhalin has been suggested to result in disruption of the membrane
dystrophin-glycoprotein complex in the cardiomyopathic hamster BIO 14.6
strain(14, 50) . We also noted the absence of adhalin
in the CHF 147 hamster heart as early as day 30, prior to the onset of
any symptoms (data not shown), while the membrane pool of dystrophin
was reduced by only 28% at much later stages (i.e. day 180) of
the disease. The expression of another membrane cytoskeletal protein,
spectrin, was also unaltered in the cardiomyopathic hamster
heart(50, 51) . While the absence of adhalin may
contribute to cardiomyopathy and myopathy in these animals, our studies
show that it is the loss of dystrophin from the cardiac myofibrils that
correlates with progression to cardiac insufficiency. We suggest that
alterations in unique cellular pools of dystrophin in cardiac muscle
may underlie some of the differential pathophysiological responses of
cardiac and skeletal muscle observed in muscular dystrophy.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
