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© The Rockefeller University Press,
0021-9525/2001//763 $5.00
The Journal of Cell Biology, Volume 153, Number 4,
, 2001 763-772
Original Article |
Alterations at the Intercalated Disk Associated with the Absence of Muscle Lim Protein
jcp{at}cell.biol.ethz.ch
In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain– containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.
Key Words: dilated cardiomyopathy • N-RAP • tropomodulin • adherens junction • gap junction
© 2001 The Rockefeller University Press
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Introduction |
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In recent years, many cardiomyopathies have been linked to mutations either in sarcomeric or in cytoskeletal proteins, resulting in familial hypertrophic cardiomyopathy and in dilated cardiomyopathy (DCM), respectively, and several mouse models have been generated to study the structural and functional changes that accompany cardiomyopathies (for reviews see Vikstrom and Leinwand 1996; Leiden 1997; Bonne et al. 1998; Seidman and Seidman 1998; Towbin 1998; Chien 1999). Both hypertrophic cardiomyopathy as well as DCM can lead to alterations in the histologic appearance of cardiac tissue, which include myocyte disarray as well as myocyte loss and fibrosis. It has been proposed that mutations in components of the force-generating apparatus tend to lead to cardiac hypertrophy, whereas changes in the force transmitting and cytoskeletal structures are characteristic of DCM (Chien 1999). So far, relatively little is known about how these mutations affect cardiomyocyte structure or the ways the cells attach to each other. Therefore, we decided to investigate cardiomyocyte and intercalated disk architecture in a mouse model for DCM, the muscle LIM protein (MLP) knockout mouse (Arber et al. 1997) and, to verify our observations on the intercalated disk structure in a second model for this disease, the tropomodulin-overexpressing transgenic (TOT) mouse (Sussman et al. 1998a).
MLP is a member of the LIM-only class of the LIM domain protein family that possesses two LIM domains. Depending on the developmental status of the cell, it can be localized in the nucleus as well as in the cytoplasm, where it is preferentially associated with actin filaments and with the Z-disk region of muscle cells (Arber et al. 1994). The two LIM domains are responsible for the differential targeting: the first LIM domain seems to be involved in nuclear targeting and the interaction with 
-actinin, a component of the Z-disk, whereas the second domain mediates association with actin filaments as well as binding to spectrin, a protein of the membrane cytoskeleton (Arber and Caroni 1996; Flick and Konieczny 2000). MLP is highly expressed during differentiation of all kinds of striated muscle, but its expression in the adult is restricted to cardiac and slow twitch fibers of skeletal muscle (Arber et al. 1994; Schneider et al. 1999). Mice that are homozygous null for MLP display symptoms of DCM with a dramatic increase in heart size (Arber et al. 1997). Two different phenotypes were described: an early postnatal phenotype that results in death by the end of the second week after birth and an adult phenotype, where the animals reach a normal age but show all the features of DCM, such as thinning of the left ventricular wall with concomitant chamber enlargement and reduced contractile function (Arber et al. 1997).
Tropomodulin is a pointed end actin-capping protein and is thought to be essential for the maintenance of thin filament length in the sarcomere (Gregorio and Fowler 1995; Sussman et al. 1998b). Alterations of its expression levels are deleterious in cultured cardiomyocytes (Gregorio and Fowler 1995; Sussman et al. 1998b) as well as in transgenic mice, where an excess of tropomodulin expression leads to the development of juvenile DCM (Sussman et al. 1998a).
Our results from the analysis of the cardiomyocyte phenotype in these mice reveal that, although sarcomere structure in MLP knockout mice appears normal in the light microscope, the intercalated disk exhibits major alterations in both mouse models for DCM: adherens junction proteins that are associated with myofibril attachment are upregulated, whereas connexin-43, the major constituent of ventricular gap junctions, is downregulated. These structural changes might provide a working hypothesis for the cause of the functional alterations that are seen in the failing heart.
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Materials and Methods |
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Antibodies
The monoclonal antibodies against myomesin (clone B4; Grove et al. 1984) and the polyclonal antibodies against myosin binding protein C (MyBP-C; Bähler et al. 1985), MLP (Arber et al. 1997), and N-RAP (Luo et al. 1997) were characterized in our laboratories. The monoclonal antibodies against sarcomeric -actinin (clone EA-53), vinculin (clone hVin), and the polyclonal antibodies against pan-cadherin, -catenin, and β-catenin were purchased from Sigma-Aldrich. The monoclonal antibody against plakoglobin (
-catenin) and the monoclonal antibody against desmoglein were from Transduction Laboratories, and the polyclonal antibody against connexin-43 was from Chemicon. The polyclonal antibody against desmoplakin (North et al. 1999) was a gift from Dr. Alison North (University of Manchester, Manchester, UK).
The secondary antibodies for immunofluorescence were bought from Jackson ImmunoResearch Laboratories (Cy3-conjugated anti–mouse and anti–rabbit Igs) and Cappel (FITC-conjugated anti–mouse and anti–rabbit Igs). Rhodamine–phalloidin was obtained from Molecular Probes, and Cy5-conjugated phalloidin was a gift from Prof. H. Faulstich (Max-Plank Institute for Medical Research, Heidelberg, Germany). Incubations with the secondary antibodies on their own gave no significant signal in either freshly isolated cardiomyocytes or semithin cryosections.
HRP-conjugated anti–mouse Igs (Dako) and anti–rabbit Igs (Calbiochem) were used for immunoblotting.
Freshly Isolated Cardiomyocytes
Muscle strips (aproximately 1 x 3 mm) from equivalent regions of the left ventricle were tied onto plastic plates at extended length and digested in 1 mg/ml collagenase (type 2; Worthington Biochemical Corp.) and 1 mg/ml verapamil (Knoll AG) in digestion solution (137 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 5.5 mM glucose, 2 mM MgCl2, 2.5 mM CaCl2, 10 mM Pipes, pH 6.5; Draeger et al. 1989) at 37°C for 90 min. After washing three times on ice with washing solution (137 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 5.5 mM glucose, 1.1 mM Na2HPO4x2H2O, 0.4 mM KH2PO4, 2 mM EGTA, 5 mM MES, 0.5 mM DTT, 100 mg/l streptomycin, pH 6.1), the pieces were cut off from the plates and placed in 500 µl cold washing solution, depending on the size of the muscle pieces. Individual cardiomyocytes were isolated by gently pipetting the digested tissue pieces up and down using a wide-mouthed Pasteur pipette. The cardiomyocytes were spun onto gelatine-coated slides using a Cytospin centrifuge (Shandon Southern), fixed for 15 min in 4% paraformaldehyde in PBS, and then permeabilized for 10 min in 0.2% Triton X-100 in PBS and incubated in a mixture of the primary antibodies diluted in 1% BSA/TBS (155 mM NaCl, 2 mM MgCl2, 2 mM EGTA, 20 mM Tris-base, pH 7.6) at 4°C overnight in a humid chamber. After washing with PBS containing 0.002% Triton X-100, secondary antibody incubations were carried out for a minimum of 3 h at room temperature (RT) to ensure complete penetration of the antibodies into the cells. After thorough washing in PBS containing 0.002% Triton X-100, the specimens were mounted in 0.1 M Tris-HCl (pH 9.5) glycerol (3:7) containing 50 mg/ml n-propyl gallate as anti-fading reagent (Messerli et al. 1993a) and sealed with nail polish.
Semithin Frozen Sections
Muscle strips were dissected from equivalent regions of the left ventricle, tied onto plastic plates, and fixed in 4% paraformaldehyde in PBS for 90 min at RT. After 10-min washes in cytoskeleton buffer (137 mM NaCl, 5 mM KCl, 1.1 mM Na2PO4, 0.4 mM KH2PO4, 4 mM NaHCO3, 5.5 mM glucose, 2 mM EGTA, 5 mM MES, 10 mg/ml streptomycin, pH 6.1) (North et al. 1994) three times, they were cut into smaller pieces and left to infiltrate with a polyvinyl pyrrolidone–sucrose mixture for 3 h on a rotating table at RT (Tokuyasu 1989). The pieces were then mounted on aluminium pins and plunge frozen in liquid nitrogen. Semithin sections (0.25 µm thick) were cut with glass knives in a Reichert Ultracryomicrotome at –70°C (Leica). The sections were retrieved on gelatine-coated coverslips, blocked first for 10 min in 0.02 M glycine/TBS, and then for 30 min in 5% preimmune goat serum in 1% BSA/TBS, and incubated in a mixture of the primary antibodies diluted with 1% BSA/TBS at 4°C overnight. After washing in 0.1% BSA in TBS five times, the secondary antibodies were applied together with Cy5-conjugated phalloidin for 1h. The coverslips were washed five times for 5 min in TBS and mounted on slides in gelvatol (Airvol 203; Air Products) containing 5 mg/ml n-propyl gallate (North et al. 1994).
Confocal Microscopy
Confocal images were recorded on an inverted microscope DM IRB/E equipped with a true confocal scanner TCS NT and a PL APO 63x/1.32 oil immersion objective (Leica) using an argon–krypton mixed gas laser. Image processing was done on a Silicon Graphics workstation using Imaris (Bitplane AG), three-dimensional multichannel image processing software specialized for confocal microscopy images (Messerli et al. 1993b).
Immunoblotting
Left ventricles were dissected, and small tissue pieces were homogenized by freeze slamming and solubilized in a modified version of SDS sample buffer (Ehler et al. 1999). Equivalent amounts of protein were run on 8–22%-gradient polyacrylamide minigels (Bio-Rad Laboratories) using the buffer system of Laemmli 1970. The proteins were blotted overnight onto nitrocellulose (Hybond-C extra; Amersham-Pharmacia Biotech; Towbin et al. 1979). After correct transfer had been established by Ponceau red staining (Serva), nonspecific binding sites were blocked by incubation in 5% nonfat dry milk in washing buffer (0.9% NaCl, 9 mM Tris, pH 7.4, 0.1% Tween-20) for 1 h at RT. Incubations with the primary and secondary HRP-conjugated antibodies were carried out as described previously (Ehler et al. 1999). Results from the chemiluminescence reaction were visualized on Fuji medical x-ray films. Only bands of the expected molecular weight as judged by Kaleidoscope prestained standards (Bio-Rad Laboratories) were present with all the antibodies used. The figures show representative blots performed with extracts from one individual animal; the experiments were repeated at least two times with extracts from at least three different animals.
Electron Microscopy
Tissue pieces from equivalent regions of the left ventricle were tied onto plastic plates, washed for 5 min in 0.1 M sodium cacodylate buffer (SCB), pH 7.2, and fixed in 2% glutaraldehyde (Fluka; Buchs) in 0.1 M SCB overnight at 4°C. After washing three times in SCB, the samples were postfixed for 1 h in 1% osmium tetroxide in SCB, dehydrated stepwise to 70% ethanol, and stained en bloc for 1 h in 2% uranyl acetate in 70% ethanol. After dehydration was completed, the cells were infiltrated with an epoxy resin based on Epon812 (Fluka) in a series of ascending mixtures of epoxy resin/propylene oxide. The specimens were then imbedded in rubber moulds filled with 100% resin and heat polymerized for 48 h at 60°C. Ultrathin sections were cut on a Reichert Diatom ultramicrotome (Leica), retrieved on carbon and Formvar-coated copper grids (Provac AG) and stained with 2% uranyl acetate in 95% methanol and Reynolds lead citrate. Preparations were examined in a JEM100C transmission electron microscope at 80 kV.
Solid Phase Binding Assays
N-RAP fragments were expressed in Escherichia coli and purified as described previously (Luo et al. 1999). Rat MLP was expressed as a histidine-tagged protein (Arber and Caroni 1996) and purified by the same methods using a plasmid containing the entire MLP sequence cloned into the BamH1 and HindIII sites of the pQE-9 vector (QIAGEN). Wells from Nunc MaxiSorp microtiter plates (Nalge Nunc) were coated with 100 µl of purified recombinant N-RAP fragments at a concentration of 0.1 µM in 6 M urea, 50 mM Tris-HCl (pH 8.0), 5 mM EGTA, and 10 mM DTT overnight at 4°C. After blocking for 1–3 h at 37°C with 0.5% BSA in PBS-T (PBS + 0.2% Tween-20), the wells were incubated overnight at 4°C with varying concentrations of purified MLP in overlay buffer (100 mM KCl, 50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 2 mM MgCl2, 2 mM ATP, 0.3 mM DTT, 0.2% Tween-20). After four washes with PBS-T, the wells were incubated with the polyclonal anti-MLP antibody diluted 1:2,000 in PBS-T + 0.5% BSA for 1 h at RT followed by another four washes. As secondary antibodies, HRP-conjugated anti–rabbit Igs (Amersham Pharmacia Biotech) were applied for 1 h at a dilution of 1:2,000 in PBS-T + 0.5% BSA, and after four washes, the color reaction was performed by incubating the wells for 30 min with 100 µl of substrate solution (0.1 mg/ml 3,3',5,5'-tetramethylbenzidine dihydrochloride, 0.01% H2O2, and 0.1 M sodium acetate, pH 5.2). The reaction was stopped by addition of an equal volume of 1 M H2SO4, and the color reaction was analyzed in an ELISA plate reader (Dynatech) by measuring the absorbency at 450 nm. Each data point represents the measurement of triplicate or quadruplicate wells. The data were analyzed as previously described (Luo et al. 1999).
Image Analysis
Evaluation of the protein expression levels as analyzed by immunoblotting as well as the evaluation of the degree of complexity of the intercalated disk in the electron micrographs were carried out using NIH Image, followed by statistical analysis in KaleidaGraph (Synergy Software).
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-actinin (Fig. 1E and Fig. F), an integral component of the Z-disk, and of myomesin (Fig. 1G and Fig. H), an M-band–associated protein, were indistinguishable in wild-type and MLP–/– cardiomyocytes (Fig. 1E and Fig. G, and Fig. F and Fig. H, respectively). Therefore, sarcomere structure appeared to be normal in MLP–/– cardiomyocytes, as judged by light microscopy.
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-catenin, +97%; E, vinculin, +51%; and H, N-RAP, +152%), whereas connexin-43, the major gap junctional protein in the ventricle, appeared downregulated in MLP–/– hearts (Fig. 3 F, –34%). The expression of desmosomal proteins like desmoplakin and desmoglein was not significantly affected (Fig. 3G and Fig. H, +4.5% and +3%, respectively). Therefore, the absence of MLP seems to result in an altered protein composition of the intercalated disks with an upregulation of adherens junction–associated proteins and a downregulation of gap junction proteins.
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Electron microscopy revealed a severe alteration of intercalated disk ultrastructure in MLP–/– and TOT hearts. The increase in width for staining of adherens junction–associated proteins as seen by immunofluorescence microscopy is not due to an increased thickness of the protein coat itself but rather to a higher degree of convolution of the membrane at the intercalated disk in MLP–/– hearts (Fig. 5 B) compared with wild type (Fig. 5 A), thus giving the impression of a broader stained region at the level of the light microscope. Random measurements of ID membrane length revealed that MLP–/– IDs showed an increase of 21.0 ± 0.2% (SD) in membrane length per standardized distance measured normal to the long axis of the cell compared with wild type. Analysis of the number of membrane loops per micrometer revealed an increase as well with 2.3 ± 0.2 (SD) loops for the MLP–/– compared with 1.5 ± 0.2 (SD) loops for the wild type, again pointing out a higher degree of convolution. Gap junctions (Fig. 5 B, arrowheads) were only rarely detected in randomly chosen fields of the sections of MLP–/– hearts, consistent with the drastically reduced staining for connexin-43 in the immunofluorescence experiments. In addition to the more irregular appearance of the IDs, detachment of neighboring cardiomyocytes was also occasionally observed (data not shown). Therefore, the increase in stained ID area as for example in the vinculin staining in Fig. 2 or in the β-catenin staining in Fig. 4 C might be explained by the broader area that is covered by the intercalated disk due to the higher degree of convolution. The same alteration, namely a more convoluted plasma membrane, can be observed when intercalated disk structure is analyzed in hearts from TOT mice (Fig. 5 C).
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Discussion |
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In addition to the changes in costamere organization, major alterations were observed at the specific cell–cell contacts of cardiomyocytes, the intercalated disks. The ultrastructural alterations observed in the electron microscope with a higher degree of convolution of the membrane and even membrane detachment are quite reminiscent of intercalated disks observed in animals with muscular dystrophy, which can also show symptoms of DCM, in desmin knockout mice that develop cardiomyopathy or in samples from old animals (Forbes and Sperelakis 1972, Forbes and Sperelakis 1985; Thornell et al. 1997).
The importance of an interplay in the stoichiometry of intercalated disk–associated proteins has been demonstrated previously, e.g., in the plakoglobin knockout mouse (Ruiz et al. 1996). Mice that are homozygous null for plakoglobin, which can be found both in desmosomes and adherens junctions, die around embryonic day 12 due to a heart defect. So far, little attention has been paid to the expression levels of adherens junction–associated proteins in cardiac disease. The organization of these proteins changes during development, since at the time of birth, most intercalated disk–associated proteins are still distributed over the entire membrane of the cardiomyocytes and restriction to the bipolar end is only achieved postnatally (Peters et al. 1994; Angst et al. 1997). In the case of the inbred hamster strain Bio14.6, which shows histological and functional symptoms similar to hypertrophic cardiomyopathy (Sakamoto et al. 1997), disruption of cell–cell adhesion with reduced expression levels of A-CAM was reported (Fujio et al. 1995). Our studies indicate that the intercalated disk phenotype that we observe might be specific for DCM since we were unable to detect an upregulation of adherens junction–associated molecules in rodent models that develop hypertrophic cardiomyopathy, such as spontaneously hypertensive rats (Ehler, E., and J.-C. Perriard, unpublished observation).
It is still unclear whether a reduced expression of connexin-43 might be a general feature of cardiac disease. Although this phenomenon has been described in hearts of patients after infarction (Smith et al. 1991; Severs 1995), in hypertrophied human hearts (Peters et al. 1993) and in animal models for cardiomyopathies (e.g., the congestive heart failure stage in a chronic aortic stenosis model in guinea pig; Wang and Gerdes 1999), there are examples of a dispersion of gap junctions from the intercalated disks rather than a downregulation of expression levels. This was found in cases of hypertrophic cardiomyopathy in humans (Sepp et al. 1996) and in the Syrian hamster strain UM-X7.1, which develops a progressive cardiomyopathy with dilation and also shows reduced levels of connexin-43 at the intercalated disks, although connexin-43 expression levels seemed to be unaffected (Luque et al. 1994). The etiology of the hypertrophy might be important as well, since during the early hypertrophic response to renovascular hypertension, a marked upregulation of connexin-43 was observed (Peters 1996). Our results with decreased levels of connexin, as shown by immunoblotting, suggest that in this case reduced amounts of protein are available that might account for the scarce occurrence of gap junctions, as seen in the electron microscope. This downregulation is not compensated by the upregulation of other connexin isoforms like connexin-40, since similar expression levels were found in wild-type and MLP–/– ventricles for this protein (data not shown). Reduced electrical communication between cardiomyocytes might result in an imbalance of free Ca2+ levels. The importance of Ca2+ cycling in cardiac disease was recently demonstrated by Minamisawa et al. 1999. A double knockout of phospholamban, the inhibitor of the muscle-specific sarcoplasmatic reticulum Ca2+ ATPase, and of MLP in mice was not only able to restore normal Ca2+ handling and thus contractility, but showed also a rescued phenotype of the cardiac cytoarchitecture (Minamisawa et al. 1999). Preliminary experiments in our group have indicated that this improvement of function goes hand in hand with the disappearance of the intercalated disk alterations as seen in the single MLP knockout mice (Ehler, E., M. Hoshijima, M. Minamisawa, K. Chien, and J.-C. Perriard, unpublished observations).
Our observations on the MLP knockout mice suggest that the biological role of MLP is to act as a stabilizing factor for myofibril attachment both at the lateral membranes and at the intercalated disk. Recently, it was reported that MLP can serve as a bridging molecule between -actinin and spectrin, thus providing a link between the myofibril and the membrane cytoskeleton (Flick and Konieczny 2000). In this respect, it is interesting to note that in mdx mice, a mouse model for muscular dystrophy, the membrane cytoskeleton of slow twitch fibers that continue to express MLP in the adult (Schneider et al. 1999) is not as severely affected as that of fast twitch fibers (Williams and Bloch 1999). A slight protective effect of MLP can also be deduced from the fact that TOT mice show an upregulation of N-RAP expression later than do MLP–/– because they still express MLP, although at reduced amounts in the adult. A decrease in MLP expression might be a general feature of late stage cardiomyopathy since it was also demonstrated in human patients with chronic heart failure and in rats with right ventricular hypertrophy induced by chronic pressure overload (Zolk et al. 2000; Ecarnot-Laubriet et al. 2000). How the feedback mechanism between expression levels of MLP and N-RAP works is completely unclear at the moment. Nevertheless, our results suggest that an upregulation of N-RAP expression can be regarded as the earliest marker for a developing DCM.
N-RAP binds actin, vinculin, and talin (Luo et al. 1999), as well as MLP (this report), and has been hypothesized to be an essential link between the terminal actin filaments of myofibrils and the complex of proteins linking these structures to the cell membrane (Luo et al. 1997, Luo et al. 1999). Our results show that a disturbance of the molecular stoichiometry of cytoskeletal proteins, such as loss of MLP or upregulation of tropomodulin, leads to an early increase in N-RAP expression. At the same time, the distribution of N-RAP becomes broader at the light microscope level and the intercalated disks become more convoluted. These changes may be an adaptive response aimed at strengthening the link between the myofibrils and the membrane. It remains to be established whether this altered intercalated disk phenotype is a general feature of DCM by investigating other animal models for this disease as well as human patients. In addition, it will be necessary to establish whether an upregulation of N-RAP expression can be used as an early marker for DCM in a clinical setting.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation grants 31.52417/97 to J.-C. Perriard and 31.40485.94 to H.M. Eppenberger, and by the research grant Cellular and Molecular Cardiology from the Union Bank of Switzerland, Zurich, Switzerland to H.M. Eppenberger. Research on the TOT mice is supported by a National Institutes of Health award (HL58224) as well as American Heart Association Awards Established Investigator and Grant-in-Aid awards to M. Sussman. Research at the Friedrich Miescher Institute (to P. Caroni) is supported by the Novartis Research Foundation.
Submitted: 16 October 2000
Revised: 27 March 2001
Accepted: 29 March 2001
Abbreviations used in this paper: DCM, dilated cardiomyopathy; MLP, muscle LIM protein; RT, room temperature; SCB, sodium cacodylate buffer; TOT, tropomodulin-overexpressing transgenic.
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