Mice that are deficient for MLP display a phenotype that is very similar to the human disease DCM, including enlargement of all four chambers of the heart, wall thinning, and reduced left ventricle function, as shown by echocardiography (Arber et al. 1997
). To characterize the alterations in cardiomyocyte cytoarchitecture during the development of DCM, we analyzed the structure of freshly isolated cardiomyocytes obtained from adult MLP−/−
mice. As shown previously, cardiomyocytes from MLP−/−
are characterized by a more irregular overall shape than their wild-type counterparts (Arber et al. 1997
). To investigate whether these changes of morphology are also reflected by changes in myofibril structure and composition, we looked at the distribution of several components of the sarcomere in freshly isolated cardiomyocytes. Although the myofibrils themselves run in a much more irregular way in MLP−/−
compared with wild-type cardiomyocytes, no gross alterations of sarcomere structure were observed. There was no effect on either thick filament structure, as shown by staining for myosin-binding protein C, which is localized in double bands in the A-band ( and ) or thin filament structure, as shown by phalloidin, which stains F-actin in the I-bands ( and ). The localization pattern of α-actinin ( and ), an integral component of the Z-disk, and of myomesin ( and ), an M-band–associated protein, were indistinguishable in wild-type and MLP−/−
cardiomyocytes ( and , and and , respectively). Therefore, sarcomere structure appeared to be normal in MLP−/−
cardiomyocytes, as judged by light microscopy.
Figure 1 Sarcomeric organization is normal in freshly isolated cardiomyocytes from MLP−/− mice. Confocal microscope images of freshly isolated cardiomyocytes from wild-type (A, C, E, and G) and MLP−/− mice (B, D, F, and H) stained (more ...)
However, alterations can be observed when the distribution pattern of vinculin is analyzed by confocal microscopy in freshly isolated cardiomyocytes. Vinculin is a component of both intercalated disks and costameres, where myofibrils are anchored laterally to the membrane and via integrins to the extracellular matrix (Pardo et al. 1983
). displays single confocal sections through an individual freshly isolated cardiomyocyte from a wild-type (A, C, and E) or a MLP−/−
mouse (B, D, and F) taken at three different levels: the substrate (A and B), through the middle of the cell (C and D), and from the top (E and F). Although wild-type cardiomyocytes displayed perfectly aligned costameres, a disturbed pattern was detected in the top (B) and bottom (F) optical sections in cardiomyocytes from MLP−/−
mice. In wild-type cardiomyocytes, vinculin molecules were organized in a somewhat parallel pattern along the intracellular side of the plasma membrane ( and ). A similar parallel pattern was seen in optical sections taken from the middle of cardiomyocytes from wild-type and MLP−/−
mice, indicating normal Z-disk association of vinculin (Terracio et al. 1990
). However, in regions underneath the plasma membrane in cardiomyocytes from MLP−/−
mice, vinculin seemed to be localized in dots that were distributed irregularly, and the parallel striations were partially lost ( and ). In addition, the freshly isolated MLP−/−
cardiomyocytes were more irregularly shaped than the rod-shaped wild-type cardiomyocytes. These irregularities were particularly prominent at the remnants of the intercalated disks of the MLP−/−
cardiomyocytes, which exhibited vinculin staining that was more conspicuous compared with wild-type cardiomyocytes. Our observations suggest that both cell–matrix as well as cell–cell contact structures are affected in cardiomyocytes from MLP−/−
Figure 2 The organization of the costameric protein vinculin is disturbed in MLP−/− cardiomyocytes. Single confocal sections of freshly isolated cardiomyocytes from wild-type (A, C, and E) and MLP−/− mice (B, D, and F) stained with (more ...)
To analyze whether similar alterations could be observed for other components of the intercalated disk, we compared the expression levels of several intercalated disk–associated proteins on immunoblots of whole muscle samples from the ventricles of wild-type and MLP−/− mice. The expression levels of all investigated adherens junction–associated proteins were elevated, as shown in (A, cadherin, +82%; B, β-catenin, +34%; C, plakoglobin; +29%; D, α-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 ( F, −34%). The expression of desmosomal proteins like desmoplakin and desmoglein was not significantly affected ( and , +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.
Figure 3 Expression levels of intercalated disk proteins are changed in adult MLP−/− hearts. Immunoblots on whole heart muscle samples of wild-type (WT) or MLP−/− mice with antibodies against cadherin (A), β-catenin (B), (more ...)
Immunofluorescence analysis of semithin cryosections of left ventricular tissue of wild-type (, A′, B, B′, G, G′, H, and H′) and MLP−/− mice (, ′, D, D′, I, I′, J, and J′) that were stained for different components of the intercalated disk together with Cy5-phalloidin to visualize F-actin (′–L′) revealed differences in the organization of the intercalated disk at the level of the light microscope. When the localization of β-catenin, for example, was compared between wild-type and MLP−/− hearts, it was apparent that not only more intercalated disks were stained, reflecting the irregular morphology with the increased number of cell–cell contacts, but that also the signal at the intercalated disk itself was broader in MLP−/− than in wild type (, compare C with A). In sections stained for the gap junction component connexin-43, a marked reduction was apparent in MLP−/− hearts ( D). The signal for desmosomes, as identified with a desmoplakin antibody, seemed indistinguishable between wild type and MLP−/− ( and ). Interestingly, when the localization of N-RAP, an intercalated disk–associated LIM domain protein was analyzed, a duplication of the N-RAP signal was invariably found in the MLP−/− hearts. Although in the wild type only a single band was stained at the intercalated disk ( H), comparable to the signal obtained for the β-catenin antibody or for vinculin (data not shown), in the case of MLP−/−, a doublet for N-RAP was always observed, associated with more pronounced actin staining ( J).
Figure 4 Changes of intercalated disk morphology as seen by immunofluorescence. Semithin cryosections of wild type (A, A′, B, B′, G, G′, H, and H′); MLP−/− (C, C′, D, D′, I, I′, J, (more ...)
To investigate whether this intercalated disk phenotype might be a general hallmark of DCM, we analyzed intercalated disk composition in a second mouse model for this disease, the TOT mouse. TOT mice show cardiomyopathic alterations within one month after birth and develop DCM with compromised contractile function and impaired myofibril organization (Sussman et al. 1998a
). In addition, the overall shape of freshly isolated cardiomyocytes from TOT mice is less regular than that from their wild-type counterparts, comparable to the morphology of MLP−/−
cardiomyocytes (data not shown). Interestingly, similar observations on intercalated disk organization as for MLP−/−
mice could also be made on semithin cryosections from TOT hearts (, ′, F, F′, K, K′, L, and L′). Adherens junction–associated proteins such as β-catenin showed an increased signal ( E), whereas the expression levels of connexin-43 seemed to be reduced drastically ( F), and the expression of the desmosomal protein desmoplakin was unchanged ( K). Additionally, in TOT mice the duplication of the N-RAP signal could be observed as well ( L), suggesting that this change in N-RAP localization might be a general feature of DCM. Thus, two mouse models of DCM with different etiologies show marked alterations of intercalated disk protein composition. The expression of adherens junction–associated proteins is upregulated, there is a reduced number of gap junctions, and a broader distribution of the intercalated disk–associated LIM domain protein N-RAP is observed.
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 ( B) compared with wild type ( 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 ( 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 or in the β-catenin staining in 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 ( C).
Figure 5 Electron microscopy of the altered intercalated disk morphology in MLP−/− hearts. Negatively stained resin sections of wild-type (A), MLP−/− (B), and TOT hearts (C). Compared with wild-type, the intercalated disks of MLP (more ...)
Since all the investigations described above were carried out in adult animals that had already developed DCM, we wanted to know whether the altered composition of the intercalated disk can be observed earlier in development, when heart function is still more or less normal. Immunoblots on SDS samples of left ventricles taken at different developmental stages are shown in ; we compared samples from newborn mice (, lanes 1 and 2), from juveniles (3 wk, lanes 3 and 4) and from adults (3–5 mo, lanes 5 and 6). The results showed that changes in expression levels of most ID-associated proteins were only apparent at the adult stage, when the phenotype of DCM is already well developed (, B–D and F). Therefore, the upregulation of adherens junction–associated proteins and the downregulation of connexin-43 seems to be a secondary effect that follows impaired function of the cardiomyocytes. In contrast, N-RAP exhibited increased expression at earlier times. In the case of MLP−/− mice, an upregulation of N-RAP expression could already be detected at birth ( E); in the case of the TOT mice, this became only obvious by the juvenile stage ( G). This suggests that elevated expression of N-RAP might serve as an early marker for DCM, even before the alterations in the expression levels of other intercalated disk–associated proteins become apparent.
Figure 6 Changes in expression levels of intercalated disk proteins during postnatal development. Immunoblots of whole heart muscle samples of wild-type (WT) and MLP−/− mice as well as of TOT of different developmental stages (newborn, lanes 1 (more ...)
N-RAP is a recently characterized nebulin-related protein that possesses a LIM domain and is concentrated at the intercalated disks in heart and at myotendinous junctions in skeletal muscle (Luo et al. 1997
, Luo et al. 1999
; Herrera et al. 2000
). Since LIM domains can act as protein–protein interaction interfaces (Schmeichel and Beckerle 1994
) and both MLP (Arber et al. 1997
) and N-RAP (Luo et al. 1997
) are concentrated at intercalated disks, we investigated whether MLP and N-RAP can directly bind to each other. Different fragments of N-RAP containing either most of the nebulin-related super repeats (N-RAP-SR), the NH2
-terminal half of the protein including the LIM domain (N-RAP-NH) or the NH2
-terminal part without the LIM domain (N-RAP-IB) were recombinantly expressed in E. coli
, and their interaction with full-length MLP was monitored in a solid phase binding assay. Strong interaction with MLP was detected for both N-RAP-IB and N-RAP-NH (). However, since MLP binding to the fragment containing the LIM domain was no greater than to N-RAP-IB, the interaction between these two proteins is probably not mediated by the N-RAP LIM domain. The absence of MLP in MLP−/−
mice might lead to increased stress at the site of the intercalated disk, leading to an upregulation of N-RAP expression. Further evidence for the importance of a feedback between MLP and N-RAP expression comes from the observation that in adult TOT mice, which show an upregulation of N-RAP, expression of MLP was markedly downregulated ().
Figure 7 Interaction between MLP and N-RAP in a solid phase binding assay. Significant binding of recombinantly expressed full-length MLP can be observed to N-RAP-IB (the region between the LIM domain and the nebulin-like repeats) as well as to N-RAP-NH (N-RAP-IB (more ...)
Figure 8 MLP expression is downregulated in hearts from adult TOT mice. Immunoblots on ventricular samples from adult TOT mice (right) and their respective wild-type strain (left) with antibodies specific for MLP reveal a decrease in MLP expression at this developmental (more ...)