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1 Division of Cardiology, Department of Medicine, and 2 Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0613
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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A mutation in the 
-sarcoglycan (SG) gene with absence
of -SG protein in the heart has been identified in the BIO14.6
cardiomyopathic (CM) hamster, but how the defective gene leads to
myocardial degeneration and dysfunction is unknown. We correlated left
ventricular (LV) function with increased sarcolemmal membrane
permeability and investigated the LV distribution of the
dystrophin-dystroglycan complex in BIO14.6 CM hamsters. On
echocardiography at 5 wk of age, the CM hamsters showed a mildly
enlarged diastolic dimension (LVDD) with decreased LV percent
fractional shortening (%FS), and at 9 wk further enlargement of LVDD
with reduction of %FS was observed. The percent area of myocardium
exhibiting increased membrane permeability or membrane rupture,
assessed by Evans blue dye (EBD) staining and wheat germ agglutinin,
was greater at 9 than at 5 wk. In areas not stained by EBD,
immunostaining of dystrophin was detected in CM hamsters at sarcolemma
and T tubules, as expected, but it was also abnormally expressed at the
intercalated discs; in addition, the expression of 
-dystroglycan was
significantly reduced compared with control hearts. As previously
described, 
-SG was completely deficient in CM hearts compared with
control hearts. In myocardial areas showing increased sarcolemmal
permeability, neither dystrophin nor -dystroglycan could be
identified by immunolabeling. Thus, together with the known loss of
-SG and other SGs, abnormal distribution of dystrophin and reduction
of -dystroglycan are associated with increased sarcolemmal
permeability followed by cell rupture, which correlates with early
progressive cardiac dysfunction in the BIO14.6 CM hamster.
-sarcoglycan; dystrophin-dystroglycan complex; heart failure; Evans blue dye
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NUMEROUS STUDIES on muscular dystrophy have focused on
dystrophin with its associated membrane-glycoprotein complex and their pathophysiological role (see Ref. 3 for review). The transmembrane dystrophin-glycoprotein complex (DGC) serves as a link between the
intracellular cytoskeleton and the extracellular matrix, connecting intracellular F-actin, dystrophin, the transmembrane sarcoglycans, and
the dystroglycans (-, -subunits) with extracellular
laminin-2. The sarcoglycan complex of
dystrophin-associated glycoproteins is composed of four subunits (,
, 
, ), sarcospan, the dystrobrevins, and the syntrophins (3,
24). Although the precise function of these components has not yet been
elucidated, the main current hypothesis regarding the primary role of
the DGC holds that it has a mechanical function to strengthen the
plasma membrane during muscle contraction (3). Also, several lines of
data support a role for dystrophin in regulating certain signal
transduction pathways, along with the syntrophins and/or calmodulin
(24). The function of the sarcoglycan complex is not known, although the sarcoglycan-sarcospan complex is assumed to serve as a molecular stabilizer of the DGC because mutations in any sarcoglycan subunit lead
to a concomitant loss or reduction of all four sarcoglycans and
sarcospan, manifested in various types of muscular dystrophy or
cardiomyopathy (24). In addition, the molecular function of each
subunit can differ (4); for example, deficiencies of - and
-sarcoglycan are associated with a cardiomyopathy, whereas -sarcoglycan deficiency is not (5).
The BIO14.6 cardiomyopathic (CM) hamster, an autosomal recessive
strain, recently has been found to have a mutation in the -sarcoglycan gene (19). Sakamoto et al. (22) subsequently reported
that two other strains of cardiomyopathic hamsters (UMX7.1 and TO-2)
also possess a mutation in the -sarcoglycan gene. In addition,
-sarcoglycan was reported to be involved in type F limb girdle
muscular dystrophy (18). Deficiency of -sarcoglycan leads to a
concomitant loss of the other sarcoglycan subunits (, , ),
sarcospan, and -dystroglycan (25). Holt et al. (9) have recently
shown that transfer of the normal human -sarcoglycan gene can
restore membrane structure and function in skeletal muscle of the
BIO14.6 hamster.
The BIO14.6 hamster develops severe cardiomyopathy with hypertrophic
features and heart failure but manifests a comparatively mild skeletal
muscle phenotype. In dystrophin-deficient mdx mice, apoptosis appears
to play a role in the onset of skeletal muscle degeneration (16, 27),
whereas in one strain of CM hamster with heart failure (CHF147) we (8)
have found that apoptosis plays a minor role compared with necrosis in
the early deterioration of left ventricular function during the first 4 wk of life. Others (20) have reported a correlation
between the degree of muscle activity and sarcolemmal damage in
skeletal muscle of mdx mice. However, the correlation between changes
in sarcolemmal permeability and left ventricular (LV) function has not
been quantified. Therefore, we investigated changes in cardiac function
and membrane permeability, as well as the subcellular distribution of
dystrophin and -dystroglycan, during early progression of the
cardiomyopathy in the BIO14.6 CM hamster.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Four-week-old normal male hamsters (F1B strain) and age-matched male BIO14.6 CM hamsters were obtained from BIO Breeders (Fitchburg, MA) and housed under controlled temperature and humidity conditions during the serial echocardiographic study. The experimental protocol was approved by the Animal Subjects Committee of the University of California San Diego, and all procedures were conducted in accordance with institutional guidelines.
Echocardiography. Ten normal and eleven BIO14.6 CM hamsters underwent serial transthoracic echocardiographic examinations at 4, 6, 8, 10, 12, and 14 wk of age to assess LV size and function. The echocardiographic methods have been described in detail in the rat (10) and mouse (26). The CM hamsters showed normal cardiac function at 4 wk of age and subsequently gradually manifested LV dysfunction; we selected 5 and 9 wk of age to correlate changes of membrane permeability and immunostaining with echocardiographic findings. Briefly, after anesthesia with pentobarbital sodium (65 mg/kg ip for normal hamsters and 55 mg/kg ip for BIO14.6 hamsters), the anterior chest was shaved, and small needle electrodes for recording the electrocardiogram were inserted into the upper two extremities and lower left extremity. Using the left lateral position, we placed the echocardiographic probe on the surface of the chest and left ventricular end-systolic and end-diastolic dimensions (LVSD, LVDD, respectively), and the thicknesses of the LV posterior wall and interventricular septum (PWT, IVST) were measured from the LV M-mode tracing. Ejection time was estimated from the aortic Doppler flow signal at the LV outflow tract. Variables derived from the echocardiographic data, including percent fractional shortening (%FS) of the LV diameter, the percent thickening of the posterior wall (%WT), mean circumferential fiber shortening rate (VCF) corrected by the R-R interval, and the estimated LV mass index, were calculated as previously described (26).
In vivo assessment of membrane permeability.
To assess changes in sarcolemmal membrane permeability of cardiac
myocytes, in vivo Evans blue dye (EBD) distribution was assessed. EBD
is a nontoxic, low-molecular-weight dye (mol wt 960) that binds to
serum albumin when infused intravenously and allowed to recirculate
(16). EBD cannot cross the sarcolemma of intact myocytes, but if the
membrane is ruptured, it is found in the intracellular space where it
binds to intracellular proteins (16). Intracellular staining by EBD
therefore identifies increased membrane permeability or sarcolemmal
rupture. We applied this method to assess membrane permeability of
cardiac myocytes in five normal and five CM hamsters at 5 and 9 wk of
age. The hamsters were anesthetized with an intraperitoneal injection
of pentobarbital sodium (65 mg/kg). Through a small incision in the
left inguinal region, the femoral vein was exposed and cannulated with
PE-50 tubing (Becton-Dickinson, Parsippany, NJ). Two percent EBD
(Sigma, St. Louis, MO) solution was dissolved in PBS (pH 7.2) and
sterilized by passage through a membrane filter with a pore size of 0.2 µm. EBD was then injected intravenously (50 µl/10 g body wt) and
the skin was sutured; the hamsters were then allowed to recover. Forty eight hours after EBD injection, an overdose of pentobarbital sodium
(100 mg/kg) was given, and the heart was quickly removed and arrested
in modified Krebs-Hensleit buffer containing high potassium chloride
(10 mg/ml). The LV was excised and divided with a cut parallel to the
atrioventricular groove, and the apical two-thirds were embedded with
OCT compound (Miles, Elkhart, IN) and frozen in isopentane chilled with
liquid nitrogen. Consecutive sections were made from each frozen heart
with the use of a cryostat (Microm, Heidelberg, Germany) with a chamber
temperature of 
25°C. Several pairs of cross-sectional
10-µm-thick adjacent sections were randomly selected from the
consecutive sections. To visualize the sarcolemma and transverse
tubules, sections were treated with 4% paraformaldehyde for 30 min and
then stained with wheat germ agglutinin conjugated to fluorescein
isothiocynate (WGA-FITC) and coverslipped with anti-fading media
(Gelvatol, Air Products and Chemicals, Allentown, PA). This section was
observed under a fluorescent microscope (HFX-DX, Nikon, Japan) equipped
with blue and green activation filters (488 and 546 nm, respectively); different barrier filters (520 and 590 nm, respectively) were used to
view and photograph WGA and EBD staining separately. Adjacent sections
were stained with hematoxylin-eosin (HE), and observed through
bright-field optics. Because heart muscle has a strong autofluorescence
in the green and red fluorescent channels, control sections were made
from EBD-free hearts treated in the same fashion and compared with
EBD-stained sections. With the use of ×100 magnification, 10 microscopic fields were randomly selected from each section, photomicrographs were taken, and the myocardial regions that were unstained and stained with EBD were scanned using a Hewlett-Packard PhotoSmart Photo Scanner. EBD-positive areas were estimated using SigmaScan Pro (Jandel Scientific Software, San Rafael, CA). The percent
area of stained myocardium was calculated from each field and averaged.
Antibodies.
Mouse monoclonal antibodies against the COOH-terminal domain of human
dystrophin (clone Dy8/6C5) and clone MANDRA-1 (kindly supplied by Dr.
G. W. Morris), the rod domain of human dystrophin (clone Dy4/6D3),
-dystroglycan (clone 43DAG1/8D5), and -sarcoglycan (clone
Ad1/20A6) were obtained from Novocastra Laboratory (Newcastle, UK). The polyclonal anti-human -sarcoglycan antibody
was kindly provided by Dr. V. E. Nigro. Secondary antibodies for light
microscopy were donkey or goat anti-mouse FITC or Cy5 (Dako) and
nanogold (Nanoprobes) conjugated to the goat anti-mouse IgG and
followed by a silver enhancement solution (Intense M, Amersham Life
Sciences, Arlington Heights, IL). Secondary antibodies for electron
microscopy were nanogold and silver enhancement solution (Amersham).
Preparation of cell lysates, immunoblotting. Normal and BIO14.6 hamster heart tissues at 6 wk were homogenized by a polytron grinder and lysed on ice for 30 min in 2 ml of ice-cold RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% deoxycholic acid, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail purchased from Sigma). Cell lysates were collected by centrifugation at 13,000 g for 10 min at 4°C. Protein concentration was determined by the Bio-Rad protein assay, using BSA as standard. Cell lysates were mixed with 6× protein sampling buffer (final concentration of buffer: 50 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 100 mM dithiothreitol, 0.0001% bromophenol), loaded on 4-20% Tris-glycine gel, and SDS-PAGE performed at 15 mA constant current. Proteins were then transferred from SDS-PAGE gels to nitrocellulose membranes at 20 V overnight. Membranes were blocked in a western buffer (0.05% Tween-20 in Tris-buffered saline, pH 7.4) with 5% skim milk for 1 h, and then incubated with primary antibodies for 2 h at room temperature. After extensive washing with Western buffer, the membranes were incubated for 1 h with horseradish-conjugated goat anti-mouse or rabbit IgG secondary antibody diluted at 1:1,000 in the Western buffer with 0.5% milk. The horseradish peroxidase-conjugated protein complex was detected by enhanced chemiluminescence according to the manufacturer's protocol (ECL kit, Amersham).
Light microscopic immunohistochemistry.
Heart tissues (n = 5) were frozen in isopentane
precooled with liquid nitrogen and sectioned at a thickness of 10 µm
on a cryostat. After air-drying for 30 min, sections were incubated for
30 min in 1% BSA/3% normal donkey or goat serum (depending on the
secondary antibody). Sections were incubated with primary antibody
(dystrophin, -dystroglycan, and -sarcoglycan) for 60 min, fixed
with 4% paraformaldehyde for 30 min, and then labeled with secondary
antibody conjugated to FITC or Cy5. Omission of the primary antibody
and omission of both primary and secondary antibodies provided negative
controls. The subcellular structure of the dystrophin-dystroglycan
complex was visualized with a laser scanning confocal microscope
(MRC-1024, Bio-Rad).
Measurement of immunolabeling intensity. For measurement of immunolabeling intensity, images were taken as eight-bit-intensity digital image (intensity value = 0-255) from each channel using the same laser excitation and photo-multiamplifier conditions used for laser scanning confocal microscopy in BIO14.6 and normal hamsters. To compare intensities of membrane immunolabeling, six cross-sectional areas were randomly chosen from multiple sections of each heart (n = 5). To determine the intensity of sarcolemma and T tubules, areas showing an intensity over the threshold (set at 25-30) were automatically detected by SigmaScan Pro (Jandel Scientific), and the average membrane intensity (arbitrary units) was computed as the average of those areas.
Electron microscopic immunohistochemistry of dystrophin. Tissue sections were fixed with 2% formaldehyde + 0.001% glutaraldehyde (n = 2) and treated with a denaturing buffer, 6 M guanidine hydrochloride in 50 mM Tris · HCl. Sections were then incubated with blocking buffer (1% BSA/3% normal goat serum/1% cold fish gelatin in 0.1 M glycine/PBS) and subsequently incubated with the primary antibody for 2 h, washed six times with PBS, and then incubated with secondary mouse IgG conjugated to 1-nm immunogold (Nanogold, Molecular Probes). After several washes in PBS, sections were fixed with 2.5% glutaraldehyde, incubated with a silver enhancement solution (Intense M), followed with 0.05% gold chloride solution, then osmicated with 1% OsO4, dehydrated, embedded into Ducarpan (ACM Resin), and then polymerized by baking. Polymerized thick sections were further sectioned onto an electron microscopy grid at a thickness of 0.1 µm. Thin sections were stained with 1% uranyl acetate and 1% lead solution (1% lead acetate, 1% lead nitrate) and observed on an electron microscope (JEM 100-CX, JEOL, Tokyo).
Statistics. Echocardiographic values are represented as means ± SD and other data as means ± SE. To compare echocardiographic data, a two-way ANOVA was used. The factors for ANOVA were the hamster strain and ages. When overall variances associated with those factors were significantly different, multiple comparison tests between normal and BIO14.6 CM hamsters, or between 4 wk and the other ages were performed using the Student-Newman-Keuls test. A P value <0.05 was regarded as significant.
To estimate significant differences between two groups of data such as EBD-stained areas in normal and BIO14.6 hamsters, unpaired t-tests were used with a P value <0.05 considered significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Echocardiographic findings.
The body weight in CM hamsters was smaller but increased significantly
with age from 4 to 14 wk in both normal and CM hamsters. The LV mass
index, estimated by echocardiography divided by the body weight, tended
to be smaller in BIO14.6 hamsters than in normals (difference
significant only at 10-14 wk of age, data not shown). As shown in
Fig. 1, the LVDD increased significantly with age in both strains of hamsters, but in the CM hamsters LVDD increased substantially more and was significantly larger than in
normal hamsters between 8 and 14 wk. The LV systolic function, measured
as %FS and corrected VCF, gradually decreased with age in CM hamsters
and was significantly reduced compared with normal hamsters between 6 and 14 wk (Fig. 1; significant between 8 and 14 wk compared with 4 wk
of same strain). The thickness of the posterior LV wall increased with
age in normal hamsters but was significantly reduced in CM hamsters
compared with normals after 8 wk (Fig. 1). All of these findings are
consistent with progression of the cardiomyopathy. The heart rates were
not significantly different between CM and normal hamsters at baseline
(448 ± 54 vs. 465 ± 45 beats/min, respectively) and did not differ
at subsequent time points.
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In vivo assessment of membrane permeability.
As shown in Fig. 2, at low and high
magnification myocytes with increased sarcolemmal permeability showed
two types of intracellular staining with EBD. In one pattern (type
A), sarcolemmal and T tubular structures could be identified by WGA
staining, and such blocks of cells exhibited a diffuse, uniform pattern
of intracellular EBD staining (Fig. 2, E and H,
asterisks) without abnormalities on the adjacent HE-stained sections
(Fig. 2B). In the second pattern (type B), the
structure of the sarcolemma and T tubules could not be identified by
WGA staining, and EBD-stained areas were irregular and less intense
(Fig. 2, E and H, pound sign). In the adjacent
HE-stained sections, clusters of necrotic myocytes with infiltration by
mononuclear or polymorphonuclear cells were observed (Fig. 2, A
and B). In normal tissues (Fig. 2C), there was no
intracellular staining with EBD, as shown in Fig. 2, F and
I.
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Immunoblotting and immunohistochemistry by light microscopy.
On immunoblot analysis, the expression of dystrophin protein was not
altered. However, -dystroglycan was reduced (~9% reduction compared with normals), and - and -sarcoglycan proteins were completely deficient (Fig. 4).
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-dystroglycan, and -sarcoglycan at 5, 9, and 14 wk of age in
normal and CM hamster hearts. -Sarcoglycan was considered
representative of all the sarcoglycans, which have been shown to be
absent or markedly reduced in the BIO14.6 hamster (25). As shown in
Fig. 5, E and F,
-sarcoglycan was found to be distributed in the sarcolemma in normal
hamster hearts, whereas it was not detected in the BIO14.6 hamsters
even at 5 wk, suggesting that the sarcoglycan complex was lost
consequent to the deficiency of -sarcoglycan. The transmembrane
component of the DGC, -dystroglycan, was localized at the sarcolemma
and T tubules as a continuous staining pattern both in normal and CM
hamster as shown in Fig. 5, A-D. However, in CM
hamster hearts the intensity of -dystroglycan was reduced by 19%
(47.03 ± 3.51 vs. 37.14 ± 1.96 arbitrary units, P < 0.05) compared with normal hearts (Fig. 5).
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-dystroglycan at the
intercalated discs of CM hamster hearts.
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-dystroglycan in these areas,
suggesting that the DGC was completely lost, despite some preservation
of cellular architecture on WGA and HE staining in these areas.
Electron microscopic immunohistochemistry of dystrophin. Because dystrophin was abnormally localized at the intercalated disc in CM hamster hearts, confirmation by electron microscopic immunolabeling of dystrophin was performed. In normal hamster hearts dystrophin was clearly distributed at the sarcolemma, frequently at the invagination site of the T tubules at the Z lines (image not shown), but was not observed at the intercalated discs. In the CM hamster hearts, dystrophin was observed in association with the sarcolemma and with the sarcolemma and the intercalated discs as observed in Fig. 6.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Relationship of membrane damage to cardiac dysfunction. By quantifying the area of in vivo intracellular staining through the use of EBD in 5- and 9-wk-old hamster hearts, an increase of regions with enhanced membrane permeability and cell damage could be observed between 5 and 9 wk. Because the development of LV dysfunction also occurred during this period, increased membrane permeability could be the initiating event leading to myocyte damage and the progression of LV dysfunction.
HE staining of type B areas of WGA and EBD staining showed fracture and loss of the myocardial cell cytoskeleton as well as large numbers of inflammatory cells, findings consistent with necrosis, whereas areas with type A staining displayed relatively mild changes in myocardial structure and few inflammatory cells. Although the fate of type A EBD-stained myocytes is uncertain, apoptosis has been reported to have a significant role in mdx skeletal muscle that stained positive with EBD (16), occurring mainly in the early period before necrosis was evident (27). However, our preliminary results in the CHF147 hamster (a strain derived from the UMX7.1 CM hamster) indicated that the contribution of apoptosis to myocardial cell damage is minor compared with that resulting from necrosis early during the onset of heart dysfunction (8). Therefore, it can be hypothesized that the continuous contraction of the heart, which is enhanced in rate and strength during exercise, may promote progressively more sarcolemmal rupture with increasing age in hearts with cardiac-sarcoglycan deficiency. The absence of dystrophin and
-dystroglycan immunolabeling in type A-positive EBD-stained
areas in which WGA staining of membrane lipid and glycoproteins were
preserved is consistent with increased sarcolemmal permeability rather
than cell rupture and suggests that loss of the DGC from the sarcolemma
predisposes to cell rupture.
Other pathological mechanisms must also be considered, including the
possibility that microvascular spasm described by Factor et al. (7)
also may be involved in causing necrosis in blocks of myocardial cells.
In this connection, the -sarcoglycan protein has been reported to be
absent in vascular smooth muscle (6, 14).
Distribution of dystrophin and -dystroglycan in CM
hamster hearts.
It was recently reported (25) that all four sarcoglycans and
-dystroglycan are lost, whereas dystrophin and -dystroglycan are
preserved in the BIO14.6 hamster heart. In the present study, we
correlated serial changes in cardiac function with the extent and type
of myocardial damage and further investigated the subcellular distribution of dystrophin and -dystroglycan in the young BIO14.6 hamster heart. In CM hamsters as young as 5 wk of age, we confirmed the
absence of -sarcoglycan, but unlike the previous study (25), we
found -dystroglycan to be reduced in the sarcolemma and T tubules.
The use of a laser scanning confocal microscope to quantify -dystroglycan may have allowed detection of the mild reduction in
cardiac muscle. We also examined the expression level of
-dystroglycan in skeletal muscle and found no significant difference
between normal and CM hamsters (data not shown), suggesting a possible contributing factor to the relatively more severe cardiac phenotype compared with that of skeletal muscle observed in BIO14.6 hamsters.
-dystroglycan. Iwata et al. (11)
reported that the subcellular distribution of dystrophin in the BIO14.6 CM hamster was not different from that in normals by using a rod domain
anti-dystrophin antibody (Dy4/6D3). We used the same clone of
anti-dystrophin antibody but found that it did not react as specifically with dystrophin in hamster tissue with immunoblotting and
immunocytochemistry as the carboxy-terminal domain antibodies (Dy8/6C5
and MANDRA-1). Both light microscopic data and electron microscopic
observations support our finding that dystrophin is abnormally
distributed at the intercalated discs in the CM hamster hearts.
Utrophin is a homolog of dystrophin localized to the intercalated discs
in heart muscle; utrophin can replace the function of dystrophin in the
dystrophin-deficient mdx mouse (21). Therefore, there is a possibility
that the anti-dystrophin antibody used in the present study might have
cross-reacted to some extent with utrophin localized at the
intercalated discs, even if it exhibits a single band on Western
immunoblotting, because the molecular weights are not distinguishable.
However, we used specific monoclonal antibodies to dystrophin, and
labeling by the antibody at the intercalated discs was absent in normal
hamster hearts where utrophin is abundant (21). Therefore,
cross-reactivity of the anti-dystrophin antibody with utrophin is a
highly unlikely explanation for our finding.
Several lines of evidence suggest that alterations are present at the
intercalated discs in the CM hamster. Kawaguchi et al. (12) reported
decreased expression of desmin and increased expression of vinculin at
the intercalated discs in CM hamsters. Luque et al. (15) described
decreased expression of the gap junction protein connexin43 on
immunohistochemical analysis in CM hamsters; connexin43 turns over
several times a day (1), and it is possible that its expression might
be affected by excessive dystrophin distribution at the intercalated
discs. However, when and how the abnormal distribution of dystrophin
occurs and its functional significance remain to be investigated. Two
possible hypotheses might be considered. Dystrophin is reported to have
interactions not only with the dystrophin-associated proteins but also
with other cytoskeletal proteins such as the integrins (28), caveolin-3 (23), and aciculin (2) in cultured skeletal myocytes. Aciculin, a cell
adhesion regulatory protein, is localized mainly at the adherens
junctions in cardiac myocytes. Because dystrophin is also localized at
the intercalated discs near the adherens junction, we can speculate
that in the CM hamster heart the cytoskeletal function of dystrophin
may be partially mediated through adherens junction proteins, such as
aciculin. Alternatively, the -sarcoglycan deficiency may cause
abnormal dissociation of dystrophin protein into the cytosol, where it
aggregates at the intercalated discs due to protein-protein
interactions, without playing any functional role.
In summary, together with the known absence of the sarcoglycans and
reduced -dystroglycan, abnormal distribution of dystrophin may
contribute to the observed loss of sarcolemmal integrity, which was
associated with early progression of cardiac dysfunction in the CM hamster.
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NOTE ADDED IN PROOF |
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Since this work was submitted, a study has been published (see Ref. 4a)
on mice deficient in -sarcoglycan showing disruption of the
sarcoglycan-sarcospan complex in vascular smooth muscle, with evidence
for a role of vascular dysfunction in the development of cardiomyopathy
in that murine model.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Tom Deerinck for excellent suggestions about electron microscopy immunolabeling, to Farid Abdel-Wahhab for technical assistance, and to Pamela Alford for manuscript preparation.
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FOOTNOTES |
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This study was supported by National Institutes of Health Specialized Center of Research in Heart Failure Grant HL-53773, an endowed chair awarded by the American Heart Association, California Affiliate, San Diego County Division (J. Ross), and by the Richard D. Winter Fund. Laser scanning confocal microscopic images and electron microscopic images were taken at the National Center for Microscopy & Imaging Research at San Diego, supported by Grant RR-04050.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Ross, Jr., Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., 0613B, La Jolla, CA 92093-0613 (E-mail: jross{at}ucsd.edu).
Received 28 June 1999; accepted in final form 19 October 1999.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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