Method for screening for cardiomyopathy

Disclosed herein are methods for screening for primary cardiomyopathy. The methods are preferably immunological methods in which the level of binding of a monoclonal or polyclonal antibody to a 50 kD glycoprotein component of a mammalian muscle tissue is determined. This level of binding is compared to the level of binding observed when non-dystrophic tissue is treated in an otherwise identical manner. A substantial reduction in the level of binding to the 50 kD glycoprotein in the experimental mammalian muscle tissue has been determined to be a screen for primary cardiomyopathy.

BACKGROUND OF THE INVENTION 
Cardiomyopathy is a generic term used to describe conditions in which 
lesions develop in the myocardium of the heart. The disease may be 
classified as secondary or primary. The secondary cardiomyopathies are 
those which are associated with an identifiable disease condition. Primary 
cardiomyopathy is a diagnosis which is made only after all known causes 
have been considered and eliminated. Primary cardiomyopathy is the most 
common form of the disease in Western countries. 
In general, there are three clinical classifications of primary 
cardiomyopathy: dilated (congestive), restrictive and hypertrophic. 
Dilated cardiomyopathy is characterized by the dysfunction of left and/or 
right ventricular function. This condition leads to cardiac enlargement. 
Restrictive cardiomyopathy is characterized by abnormal diastolic function 
associated with excessively rigid ventricular walls. Hypertrophic 
cardiomyopathy is characterized by left ventricular hypertrophy. 
Present methods available for the evaluation of the cardiomyopathies 
include chest roentgenogram, electrocardiogram, echocardiogram, 
radionuclide studies and cardiac catheterization. Although the 
characterization of the cardiomyopathies on the basis of clinical 
presentation is useful, a method that would enable an etiologic diagnosis 
is preferable. In many cases, such a diagnosis is not possible. 
SUMMARY OF THE INVENTION 
The subject invention relates to methods for detecting primary 
cardiomyopathy by immunological techniques. These methods are based on the 
discovery of a substantial reduction in the levels of a 
dystrophin-associated glycoprotein in mammalian muscle tissue samples from 
a cardiomyopathy animal model and from a human patient. 
Preferred methods for detecting levels of dystrophin-associated proteins 
are immunological methods. Particularly useful methods are fluorescence 
microscopy and immunoblot techniques. Such methods enable an etiologic 
diagnosis for cardiomyopathy and are preferable to the current 
characterization on the basis of clinical presentation. 
DETAILED DESCRIPTION OF THE INVENTION 
It has been reported that a set of proteins are tightly associated with 
dystrophin in vivo. These include, for example a dystrophin-associated 
protein having a molecular weight of 59 kD (59-DAP), and four 
dystrophin-associated glycoproteins having the molecular weights 35 kD 
(35-DAG), 43 kD (43-DAG), 50 kD (50-DAG) and 156 kD (156-DAG). Antibodies 
specifically reactive with these antigens have been identified. 
It has been demonstrated that a substantial reduction in the level of 
dystrophin and/or dystrophin-associated proteins correlates with the 
muscular dystrophy phenotype (particularly with respect to Becker's or 
Duchenne's muscular dystrophy). Based on this observation, it was of 
interest to determine whether a reduction in the level of these proteins 
could be correlated with another disease classification which is 
characterized by muscle cell necrosis. More specifically, it was of 
interest to determine whether the appearance of primary cardiomyopathy 
could be correlated with a substantial reduction in dystrophin and/or the 
dystrophin-associated proteins. 
The subject invention in based on the discovery of substantial reduction in 
the levels the 50-DAG in muscle tissue from an animal model and from a 
human patient afflicted with primary cardiomyopathy. In addition to the 
50-DAG, preliminary evidence suggests that the levels of other 
dystrophin-associated proteins are also affected. 
The invention relates to a method for diagnosing primary cardiomyopathy. A 
muscle tissue biopsy sample is obtained from the mammal for analysis 
according to the methods described herein. Preferred muscle tissue biopsy 
samples are obtained from cardiac or skeletal muscle tissue. Biopsy 
samples can be obtained using a variety of methods including, for example, 
the use of a biopsy needle. The muscle tissue is analyzed for 
substantially reduced levels of the 50-DAG. 
Substantial reduction, as used herein, refers to a reduction in the level 
of the 50-DAG in experimental tissue of at least about 50% relative to the 
level of the 50-DAG in non-cardiomyopathic tissue. Quantitative methods 
for monitoring levels of dystrophin-associated protein have been 
previously reported (see e.g., Ervasti et al., Nature 345:315-319 (1990). 
Analysis of the levels of dystrophin-associated proteins in the muscle 
tissue sample is best carried out by immunological methods. Monoclonal or 
polyclonal antibodies which bind specifically to dystrophin-associated 
proteins are contacted with the muscle tissue biopsy sample under 
conditions appropriate for the binding of antibody to antigen. 
Particularly useful for this analysis are affinity purified polyclonal 
antibodies. 
Antibodies specific for the various components of the 
dystrophin-glycoprotein complex are prepared by conventional methods. 
Methods for isolating the dystrophin-glycoprotein complex have been 
previously reported. For example, the dystrophin-glycoprotein complex can 
be isolated from detergent solubilized skeletal muscle membranes using 
affinity chromatography and density gradient ultracentrifugation. Lectins 
are proteins or glycoproteins which bind certain sugars or 
oligosaccharides. This property can be used to isolate certain 
glycoproteins from a complex mixture and is extremely useful as a general 
approach to the purification of membrane proteins, many of which are 
glycosylated. In the present invention, the linked components of the 
dystrophin-glycoprotein complex can be isolated as an intact complex with 
lectins that bind to the glycoprotein components of the complex. The 
lectins are typically coupled to a solid support such as a chromotographic 
gel (i.e., sepharose, agarose, etc.) and a complex mixture of membrane 
components is passed through a chromatography column containing the gel 
with bound lectin. The glycoproteins of membrane components bind to the 
lectin while the other components of the mixture pass through the column. 
A variety of lectins can be used in affinity-based methodologies to 
isolate the dystrophin-glycoprotein complex. 
The dystrophin-glycoprotein complex can be further purified using density 
gradient ultracentrifugation. The eluate from the affinity column as 
described above is applied as a narrow band to the top of a solution in a 
centrifuge tube. To stabilize the sedimenting components of the eluate 
against convection mixing, the solution beneath the band contains an 
increasingly dense solution of an inert, highly soluble material such as 
sucrose (a density gradient). Under these conditions, the different 
fractions of the eluate sediment at different rates forming distinct bands 
that can be individually collected. The rate at which each component 
sediments depends on its size and shape and is normally expressed as its 
sedimentation coefficient or S value. Using this technique, the size of 
the dystrophin-glycoprotein complex was estimated to be approximately 18 S 
by comparing its migration to that of standards of varying size. 
Another form of affinity chromatography which can be used to isolate the 
dystrophin-glycoprotein complex is known as immunoaffinity purification. 
This technique utilizes the unique high specificity of polyclonal and 
monoclonal antibodies as well as selected lectins. Such highly specific 
molecules are extremely valuable tools for rapid, selective purification 
of antigens. In principle, the antigen is coupled (immobilized) on a 
column support and this is used to selectively adsorb antigen from a 
mixture containing many other antigens. The antigens for which the 
antibody has no affinity can be washed away, and the purified antigen then 
eluted from the bound antibody or lectin with an elution buffer. The 
separation and isolation of the components of the dystrophin-glycoprotein 
complex can be accomplished by conventional techniques such as 
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or gel filtration high 
pressure liquid chromotography. 
Monoclonal and polyclonal antibodies specific for non-dystrophin components 
of the dystrophin glycoprotein complex are prepared by conventional 
methods. Monoclonal antibodies can be prepared, for example, by immunizing 
an animal with a preparation containing the dystrophin-glycoprotein 
complex. A fused cell hybrid is then formed between antibody-producing 
cells from the immunized animal and an immortalizing cell such as a 
myeloma to produce a hybridoma. The hybridomas are then screened for 
production of anti-non-dystrophin component antibodies using standard 
immunological techniques. 
Polyclonal antibodies can be prepared by immunizing an animal with a crude 
preparation of the dystrophin-glycoprotein complex or the purified 
non-dystrophin components of the complex. The animal is maintained under 
conditions whereby antibodies reactive with the components of the complex 
are produced. Blood is collected from the animal upon reaching a desired 
titer of antibodies. The serum containing the polyclonal antibodies 
(antisera) is separated from the other blood components. The polyclonal 
antibody-containing serum can optionally be further separated into 
fractions of particular types of antibodies (e.g., IgG or IgM). 
Additionally, polyclonal antibodies can be affinity purified to generate a 
polyclonal antibody preparation which is antigen specific. This is best 
accomplished by attaching a purified component of the 
dystrophin-glycoprotein complex to a solid support and preparing an 
affinity column. 
As shown in the Exemplification section which follows, it has been 
determined that a reduction in the extent of antibody binding to the 
50-DAG correlates with primary cardiomyopathy. A diagnostic method for the 
detection of cardiomyopathy can be carried out in a variety of formats. 
In the preferred embodiment of the diagnostic method of the invention, a 
muscle biopsy sample is treated in a procedure which renders the 
non-dystrophin components available for complexing with antibodies 
directed against said components. Muscle biopsy samples can be taken from 
any muscle; preferably cardiac or skeletal muscle. The amount of muscle 
obtained should be enough to extract the components of the 
dystrophin-glycoprotein complex from muscle membranes and detect their 
presence by the diagnostic methods described within this application. 
Alternative methods of extraction can be used. 
For biopsy samples greater than 500 mg, the muscle tissue can be 
homogenized by mechanical disruption using apparatus such as a hand 
operated or motor driven glass homogenizer, a Waring blade blender 
homogenizer, or an ultrasonic probe. Homogenization can occur in a buffer 
comprising 20 mM sodium pyrophosphate, 20 mM sodium phosphate monohydrate, 
1 mM magnesium chloride, 0.303M sucrose,, 0.5 mM EDTA, pH 7.1, with 
various protease inhibitors such as aprotinin (0.5 .mu.g/ml), benzamidine 
(100 .mu.g/ml), iodoacetamide (185 .mu.g/ml), leupeptin (0.5 .mu.g/ml), 
pepstatin A (0.5 .mu.g/ml) and PMSF (40 .mu.g/ml). Heavy microsomes can be 
prepared from homogenized skeletal muscle by the method of Mittchel, et 
al., J. of Cell Biol, 95: 1008-1016 (1983). The microsomes are then washed 
with a physiological salt solution and solubilized in saline containing 
detergent and protease inhibitors. Following solubilization of the 
microsomes, the sample is treated with sodium SDS. In the present case, 
SDS acts to dissociate the linked components of the 
dystrophin-glycoprotein complex, thereby allowing their separation. 
For muscle biopsy samples less than 500 mg, an alternative extraction 
procedure can be used. Samples are frozen in liquid nitrogen and crushed 
using a mortar and pestle and prepared for electrophoresis by treatment 
with SDS as described by Hoffman et al., (N. Eng. J. of Med. 318:1363-1368 
(1988)). 
The SDS treated sample is then electrophoresed by polyacrylamide gel 
electrophoresis (PAGE). Following separation by SDS-PAGE, the separated 
components of the dystrophin-glycoprotein complex are transferred from the 
gel matrix to a solid support to generate a protein transfer blot. 
Alternatively, tissue specimens (e.g., human biopsy samples) can be tested 
for the presence of the components of the dystrophin-glycoprotein complex 
by using monoclonal or polyclonal antibodies in an immunohistochemical 
technique, such as the immunoperoxidase staining procedure. In addition, 
immunofluorescent techniques can be used to examine human tissue 
specimens. In a typical protocol, slides containing cryostat sections of 
frozen, unfixed tissue biopsy samples are air dried and then incubated 
with the anti-non-dystrophin component antibody preparation in a 
humidified chamber at room temperature. The slides are layered with a 
preparation of fluorescently labeled antibody directed against the 
monoclonal antibody. The staining pattern and intensities within the 
sample are determined by fluorescent light microscopy. 
The antibodies of the present invention can also be used in an 
enzyme-linked immunoadsorbant assay (ELISA) for determining the absence or 
presence of non-dystrophin components of the dystrophin-glycoprotein 
complex. Antibodies against non-dystrophin components to be measured are 
adsorbed to a solid support, in most cases a polystyrene microtiter plate. 
After coating the support with antibody and washing, a solubilized sample 
is added. If a non-dystrophin component is present for which the 
antibodies are specific, they will bind to the adsorbed antibodies. Next, 
a conjugate that will also bind to the non-dystrophin component is added. 
Conjugates are secondary antibody molecules to which an enzyme is 
covalently bound. After addition of a chromogenic substrate for the 
enzyme, the intensity of the colored reaction products generated will be 
proportional to the amount of the non-dystrophin component present, 
determination of the intensity of the color produced by a standard series 
of non-dystrophin component concentrations will allow the calculation of 
the amount of non-dystrophin component in an unknown sample. Many 
variations of this assay exist as described in Voller, A., Bidwell, D. E. 
and Bartlett, A., The Enzyme Linked Immunosorbent Assay (ELISA): A guide 
with abstracts of microplate applications, Dynatech Laboratories, 
Alexandria, Va. ( 1979) and are hereby incorporated by reference. 
The invention is now further and specifically illustrated by the following 
examples. All parts and percentages are by weight unless otherwise 
specified.

EXAMPLES 
Example 1 
The inbred Syrian hamster strain Bio-14.6 is an established animal model 
for cardiomyopathy, characterized by muscle cell necrosis and hypertrophy, 
ultimately leading to congestive heart failure. In this Example, the 
status of the individual components of the dystrophin-glycoprotein complex 
in muscle tissue from control and cardiomyopathic hamsters is compared 
with that of the mdx mouse, an animal model for Duchenne muscular 
dystrophy. 
Isolation of Cardiac and Skeletal Muscle Membranes 
Total cardiac and skeletal muscle membranes were prepared from age-matched 
control hamsters and cardiomyopathic Syrian hamsters, as well as control 
and mdx mice. Control hamster strain F1B and cardiomyopathic hamster 
strain Bio-14.6 were obtained from Biobreeders, Watertown, Mass. Hind leg 
and back muscle were dissected and homogenized in 7.5 volumes of 
homogenization buffer (20 mM sodium pyrophosphate, 20 mM sodium phosphate 
monohydrate, 1 mMMgCl.sub.2, 0.303M sucrose, 0.5 mM EDTA, (pH 7.0)) by a 
Polytron PTS-10-S (Kinematic GmbH, Luzern, Switzerland) in the presence of 
a protease inhibitor cocktail. Homogenates were centrifuged for 15 minutes 
at 1100.times.g, the supernatant filtered through 4 layers of cheese cloth 
and total membranes obtained by a final centrifugation for 37 minutes at 
140,000.times.g. Protein was determined by conventional methods using 
bovine serum albumin as a standard. 
Antibodies 
Affinity-purified sheep antibodies to the individual components of the 
dystrophin-glycoprotein complex were produced and characterized as 
described by Ohlendieck and Campbell (J. Cell Biol. 115:1685-1694 (1991)). 
Antibodies against the dystrophin-associated proteins of 35-DAG, 43-DAG, 
50-DAG, 59-DAP and 156-DAG are highly specific for their respective 
antigen and do not immunologically cross-react with each other. In 
addition, the following monoclonal antibodies were used in this study: mAb 
VIA4.sub.2 to dystrophin, mAb IIH6 to dystrophin-associated glycoprotein 
of 156-DAG, mAb IID8 to cardiac sarcoplasmic reticulum Ca.sup.2+ -ATPase 
of fast-twitch skeletal muscle, mAb VIID1.sub.2 to calsequestrin, mAb 
IIID5 to .alpha..sub.1 -subunit of dihydropyridine receptor, mAb McB2 to 
Na/K-ATPase, and mAb RyR-1 to cardiac ryanodine receptor. Each of these 
antibodies have been described in printed publications. Polyclonal 
antibodies to skeletal muscle ryanodine receptor were raised in sheep and 
rabbit antibodies against the unique C-terminal sequences of human 
dystrophin and human dystrophin-related protein were affinity-purified and 
characterized. 
Results 
Immunolocalization of components of the dystrophin-glycoprotein complex in 
skeletal and cardiac muscle from normal and cardiomyopathic Syrian 
hamsters 
Transverse skeletal muscle cryosections from six-week-old FiB (Control) or 
BIO 14.6 cardiomyopathic (CMH) hamsters were stained with hematoxylin and 
eosin. Hematoxylin and eosin staining of frozen cross-sections of 
cardiomyopathic hamster (CMH) skeletal muscle revealed muscle fibers of 
various sizes with rounded contours and central nucleation. Central 
nucleation is characteristic of muscle cell regeneration. 
Additional cryosections were labeled by indirect immunofluorescence (see 
e.g., Zubrzycka-Gaarn et al., Nature 333:466-469 (1988) and Ohlendieck et 
al., J. Cell. Biol. 122:135-148 (1991)) with affinity-purified antibodies 
against the C-terminus of dystrophin (DYS) or against the 
dystrophin-associated proteins 156-DAG, 59-DAP, 50-DAG, 43-DAG and 35-DAG. 
50-DAG antibodies were affinity-purified against the entire protein or 
against a 50-DAG peptide as described by Noorgard et al. (J. Mol. Cell. 
Cardiol. 19:589-594 (1987)). Antibody binding was detected using a 
biotinylated secondary antibody (Vector Laboratories) and 
fluorescein-conjugated streptavidin (Jackson ImmunoResearch Laboratories). 
Sections from control and cardiomyopathic hamsters were placed on the same 
microscopy slide to ensure identical treatment, and photographs were 
processed using identical times and conditions for a given antibody on 
both control and cardiomyopathic sections. Indistinguishable results were 
observed using skeletal muscle obtained from 2- to 24-week-old hamsters. 
All DAGs were localized at the cell periphery of hamster skeletal muscle, 
consistent with their localization in mouse and rabbit. The 59-kDa 
dystrophin-associated protein (59-DAP) and the 156- and 43-kDa DAGs 
(156-DAG and 43-DAG) were present at apparently equal levels in normal and 
CMH muscle as determined by immunofluorescence, whereas the 35-kDa DAG 
(35-DAG) was slightly decreased at the CMH sarcolemma. However, the 50-kDa 
DAG (50-DAG) was not detected by immunofluorescence in CMH muscle using 
either of two affinity-purified antibodies against this protein. Identical 
results were obtained using tissue from hamsters ranging from 2 to 24 
weeks of age. Thus, 50-DAG was undetectable before, during, and after the 
age of primary cardiac necrosis. 
Immunohistochemical analysis of cardiac cryosections 
Immunohistochemical analysis of cardiac cryosections was performed to 
determine levels of dystrophin and DAPs in normal and CMH cardiac 
sarcolemma. Cardiac ventricle cryosections from 19-week-old FIB (control) 
or BIO 14.6 cardiomyopathic (CMH) hamsters were labeled by indirect 
immunofluorescence with affinity-purified antibodies against the 
C-terminus of dystrophin (DYS) or the DAGs as described above. 
All components of the dystrophin-glycoprotein complex were clearly 
localized to normal hamster cardiac sarcolemma. Also, staining of small 
processes leading inward from the sarcolemma was consistent with the 
presence of these proteins in hamster cardiac T-tubules as has been 
observed in rabbit cardiac muscle. Dystrophin and 59-DAP were present at 
nearly equal levels in normal and CMH cardiac membranes. 35-DAG staining 
was also lower in CMH membranes, and 50-DAG was not detected in CMH heart. 
In contrast to the findings in skeletal muscle, 156-DAG and 43-DAG 
appeared decreased in CMH cardiac sarcolemma relative to that of normal 
hamsters. Thus, DAGs appear to be more deficient in CMH cardiac muscle 
than in CMH skeletal muscle. This may explain why the cardiomyopathic 
hamsters experience more severe cardiac symptoms than skeletal muscle 
symptoms. 
Immunoblot analysis of muscle tissue homogenates 
To more accurately quantify the abundance of dystrophin and DAGs in the BIO 
14.6 hamster, immunoblot analysis was performed on skeletal muscle and 
cardiac homogenates. Tissue homogenates were prepared from control 
hamsters and cardiomyopathic Syrian hamsters as described previously 
(Ohlendieck et al., J. Cell Biol. 112:135-148 (1991)). Proteins were 
fractionated on 3-12% gradient SDS-polyacrylamide gels. Transfer of 
proteins to nitrocellulose was performed according to Towbin et al. (J. 
Natl. Acad. Sci. USA 76:4350-4354 (1979)), and immunoblot staining with 
antibodies was performed as previously described (Campbell and Kahl, 
Nature 338:259-262 (1989)). Blots were stained with monoclonal antibody 
McB2 against the Na/K-ATPase, an affinity-purified rabbit antibody against 
the C-terminal of dystrophin (DYS), monoclonal antibody IIH6 against the 
156DAG, or a mixture of affinity-purified antibodies against the 35-, 43-, 
50-, and 59-kDa DAPs (DAPs). 
Control experiments were performed on immunoblots of homogenates to 
demonstrate that any changes in protein levels between normal and 
cardiomyopathic hamsters were not due to nonspecific degradation of 
membrane proteins. The pattern of binding of wheat germ agglutinin, 
concanavalin A, and jacalin to CMH skeletal muscle and cardiac homogenates 
was unaffected, indicating that any changes in DAG levels were specific 
rather than due to general effects in necrotic tissue. Additionally, 
integral membrane proteins involved in excitation-contraction coupling 
specifically the ryanodine receptor, dihydropyridine receptor, and 
Ca.sup.2+ - ATPase were present at comparable levels in both control and 
CMH skeletal muscle and cardiac homogenates. These results indicate that 
the majority of integral membrane proteins and glycoproteins are 
unaffected in cardiomyopathic hamster skeletal muscle and heart. 
Furthermore, the equal density of Na/K-ATPase in control and 
cardiomyopathic hamster skeletal muscle and heart demonstrates that 
homogenates from both strains contain equal amounts of sarcolemma. 
In skeletal muscle, dystrophin, 156-DAG, and 59-DAP were present at 
approximately equal levels in control and cardiomyopathic hamsters. 43-DAG 
and 35-DAG were present at lower levels in CMH skeletal muscle relative to 
that of controls. However, 50-DAG was undetectable on immunoblots of 
skeletal muscle homogenates from cardiomyopathic hamsters. In heart, as in 
skeletal muscle, dystrophin and 59-DAP were present at equal levels in 
normal and cardiomyopathic hamsters, whereas 43-DAG and 35-DAG were 
reduced in CMH heart relative to normal heart. 50-DAG was also undetected 
on immunoblots of BIO 14.6 cardiac homogenates. However, 156-DAG was 
greatly reduced in CMH heart. It remains to be determined why 156-DAG is 
deficient in CMH heart but not skeletal muscle although both tissues are 
affected by the disease. 
Skeletal and cardiac myocytes of cardiomyopathic hamsters contain elevated 
levels of intracellular calcium. Increased levels of dihydropyridine 
receptors and ryanodine receptors have been reported in CMH cardiac 
membranes but not in cardiac homogenates. Using immunoblot analysis no 
differences were observed between normal and CMH skeletal muscle or heart 
in levels of Na+/K+-ATPase, dihydropyridine receptor, ryanodine receptor, 
or Ca.sup.2+ -ATPase. 
Immunoblot analysis of components of the dystrophin-glycoprotein complex in 
cardiac muscle membranes of normal and mdx mice 
The mdx mouse does not experience cardiac symptoms or histopathological 
changes in cardiac muscle. However, all DAGs are greatly reduced in 
cryosections and total membranes of mdx mouse skeletal muscle. Here 
immunoblot analysis was used to determine the status of DAGs in 
dystrophin-deficient, histologically normal mdx heart. Immunoblots from 
control and mdx murine cardiac tissue were stained with monoclonal 
antibody McB2 against the Na/K-ATPase, an affinity-purified rabbit 
antibody against the C-terminal of dystrophin (DYS), monoclonal antibody 
IIH6 against the 156-DAG, or a mixture of affinity-purified antibodies 
against the 35-, 43-, 50-, and 59-kDa DAPs (DAPs). 
The 156-, 50-, 43-, and 35-kDa DAGs were present at approximately normal 
levels in mdx cardiac membranes, but the 59-kDa DAP was greatly reduced. 
Identical results were obtained using mice ranging in age from 5 to 55 
weeks. The specific loss of 59-DAP is consistent with its direct 
association with dystrophin as has been previously proposed. The remaining 
DAGs are preserved in cardiac sarcolemma, presumably by interacting with 
dystrophin-related protein (DRP), an autosomal homolog of dystrophin that 
is present in cardiac myocytes of mdx mice. DRP expression is absent from 
cardiac muscle of normal mice. Thus, mdx mice may be relatively free of 
cardiomyopathies due to the ability of DRP to compensate for the loss of 
dystrophin by associating with membrane-spanning DAGs to preserve the link 
between cytoskeleton and extracellular matrix. 
This work demonstrates that the 50-kDa DAG is deficient in skeletal muscle 
and heart of the BIO 14.6 cardiomyopathic hamster, although dystrophin is 
present at normal levels and at its normal subcellular location. 
Example 2 
To determine whether human cardiomyopathic tissue would mimic the protein 
distribution pattern described above in connection with the animal model 
system, immunoblots were prepared from human cardiac tissue. The tissue 
samples were obtained from a patient diagnosed with primary cardiomyopathy 
and from an unaffected control patient. The immunoblot preparation, 
staining and analysis was carried out as described above. These 
experiments demonstrated a substantial reduction in the level of the 
50-DAG in the cardiomyopathic tissue when compared to the control tissue. 
As was observed in the animal model system, reduction in other 
dystrophin-associated proteins was also observed. The significance of the 
reduction in the levels of dystrophin-associated proteins other than the 
50-DAG remains to be investigated. 
Equivalents 
Those skilled in the art will know, or be able to ascertain using no more 
than routine experimentation, many equivalents to the specific embodiments 
of the invention described herein. These and all other equivalents are 
intended to be encompassed by the following claims.