The FASEB Journal • Research Communication
A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization Manolis Mavroidis, Panagiota Panagopoulou, Ioanna Kostavasili, Noah Weisleder,1 and Yassemi Capetanaki2 Cell Biology Division, Center of Basic Research, Biomedical Research Foundation, Academy of Athens, Athens, Greece. A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyoplathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.—Mavroidis, M., Panagopoulou, P., Kostavasili, I., Weisleder, N., Capetanaki, Y. A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization. FASEB J. 22, 3318 –3327 (2008)
ABSTRACT
Key Words: intercalated discs 䡠 IF proteolysis 䡠 transgenic mice Desmin, the major muscle-specific intermediate filament (IF) protein, is essential for maintaining a spatial and functional relationship between the Z discs of the contractile apparatus and mitochondria, nuclei, sarcolemma, and other membranous organelles (for review, see ref. 1 and references therein). Desmin, like all IFs, displays the typical central a-helical rod domain of 3318
highly conserved structure and sequence, flanked by nonhelical end domains. The helical rod domain is interrupted by three short nonhelical linkers, thus generating four helices, responsible for coiled-coil dimer formation. The nonhelical amino-terminal (head) and carboxy-terminal (tail) domains of IF subunit proteins are highly variable in sequence and size. Although their specific role is largely unknown, the head and tail domains are sites of post-translational modifications by phosphorylation and glycosylation, regulating dynamic aspects of IF organization and structure during cell cycle and developmental programs. The diversity of the head and tail domains and their post-translational modifications constitute the basis of the tissue-specific function of the various IF family members’ proteins. Desmin-null (des⫺/⫺) mice develop multiple defects in all muscle types (2, 3), particularly within cardiac muscle, where dilated cardiomyopathy and heart failure develop with age (4). The initiating events in des⫺/⫺ cardiomyopathy appear to be mitochondrial defects and cardiomyocyte death, accompanied by extensive calcification and fibrosis (refs. 5, 6; reviewed in ref. 7). Several different mutations in the desmin gene have been identified in families whose affected members present myopathies of varying intensity (8). Most of the mutations identified are at the central rod domain, leading to inadequate supply of functional desmin (9) as well as dominant-negative effects resulting in the disruption of a preexisting IF network (10, 11). The first mutation described at the desmin tail domain was a missense mutation (I451M) and was proposed as the genetic cause of idiopathic dilated cardiomyopathy (12). More recently, members of another family carrying the same mutation (I451M) and presenting a slowly progressive skeletal myopathy have 1
Current address: Department of Physiology and Biophysics, Robert Wood Johnson Medical School, Piscataway, NJ, USA. 2 Correspondence: Soranou Efesiou 4, 11527-Athens, Greece. E-mail:
[email protected] doi: 10.1096/fj.07-088724 0892-6638/08/0022-3318 © FASEB
been identified (13). In both families there is incomplete penetration of the mutation, with members carrying the mutation and being clinically asymptomatic. The I451M mutation is less pathogenic than previously studied dominant mutations in the rod domain of the desmin molecule. The disease mechanism of the tail domain I451M mutation presents a challenging and still unresolved problem (13). Two new mutations, Thr453Ile and Lys449Thr, associated with restrictive cardiomyopathy (14) and myofibrillar myopathy (15), respectively, are located in the same area. Although the desmin tail domain varies in length and sequence from that of other IFs, this area has a motif (IKTIETRDG) considerably conserved between type III IFs [desmin, vimentin, peripherin, and glial fibrillary acidic protein (GFAP)] and thus possibly significant. A potential role of that motif in the lateral packing of type III IFs has been suggested by in vitro studies (16). Nevertheless, the role of the type III IF tail domain remains elusive. It has been proposed that the head and tail domains protrude from fully formed IFs, allowing access to other cellular proteins (17, 18). This raises the possibility that the nonhelical domains play another role, other than filament assembly and tissuespecific stability. However, there is little direct evidence to verify that this is so. Early reports of interactions between the head domain and components of the plasma membrane (19), between the tail domain and the nuclear envelope (20), and between the tail domain and actin-containing structures (21) have had little to no follow up. Generation and study of transgenic animals carrying the I451M mutation in cardiac tissue has allowed the unraveling of a potential in vivo role of the desmin tail domain in the stabilization of desmin IFs, and in the formation of IF scaffold around Z discs, as well as the implications of the above in the onset of cardiomyopathy.
MATERIALS AND METHODS Transgenic mouse generation Desmin-null mice were previously generated in the 129SV inbred background by homologous recombination, through standard methods (2). Transgenic mutant desmin(I451M) mice were generated using the cardiac-specific ␣-myosin heavy-chain promoter (22) to drive expression of a murine desmin(I451M) cDNA in a C57BLK/6 background by pronuclear injection. Mutant desmin(I451M) cDNA was constructed by PCR, taking advantage of a unique BclI restriction site, 15 bp upstream of the mutation site. Two expressing lines, a-TgdesI451M and b-TgdesI451M, were bred with des⫺/⫺ mice to generate F1 progeny. F1 mice were bred together to generate all potential genetic permutations. The procedures for the care and treatment of animals were according to institutional guidelines which follow the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). DESMIN ILE451MET MUTATION
Immunofluorescence Excised hearts were frozen in isopentane cooled in a liquid nitrogen bath. Slides were fixed in 70% methanol/30% acetone at ⫺20°C for 10 min. Frozen sections (10 m) were incubated with anti-desmin monoclonal (DE-U-10 D1033), anti-desmin polyclonal (D8281) and anti-␣-sarcomeric actinin monoclonal (clone EA-53, A7811) antibodies, all from Sigma (St. Louis, MO, USA), at 1:10, 1:30 and 1:1000 dilutions, respectively, in 5% BSA in PBS-Tween-20 (0.02%) for 2 h at room temperature. For desmoplakin (DP) staining, the rabbit, anti-human DP polyclonal antibody (AHP320; Serotec, Kidlington, UK) was used at 1:100 dilution. Sections were incubated with secondary antibodies Alexa Fluor-568 and Alexa Fluor-488 (Molecular Probes, Leiden, The Netherlands). Images were collected on a Zeiss Axiovert 100 2TV microscope equipped with an ⫻63 NA 1.4 objective (Carl Zeiss, Oberkochen, Germany) and used for Deltavision deconvolution processing (Applied Precision, Issaquah, WA, USA) using SoftWorX software and running on a Silicon Graphics workstation (Silicon Graphics, Sunnyvale, CA, USA) (23). For confocal imaging, the Leica TCS SP5, DMI6000, microscope (inverted, with the acquisition software LAS-AF, at 23–24°C; Leica Microsystems, Wetzlar, Germany) was used. Anti-desmin antibody production A rabbit polyclonal antibody (␣-desminN15 antibody) was raised against a synthetic 15peptide (MSQAYSSSQRVSSYR) corresponding to the desmin amino terminus, coupled to preactivated keyhole limpet hemocyanin using 1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Pierce, Rockford, IL, USA) according to the manufacturer’s procedure. Preparation of cytoskeletal protein-enriched fractions: immunoblotting Cardiac tissue was homogenized in buffer A: PBS, 0.6 M KCl, 1% Triton X-100, 2 mM EDTA, 1 mM dithiothreitol (DTT) with protease inhibitor cocktail (Sigma P8340), 2mM PMSF, and phosphatase inhibitors (0.2 mM Na2VO3 and 1 mM NaF). The homogenate was centrifuged for 10 min at 3000 g. The pellet, enriched in cytoskeletal proteins, was resuspended in SDS-PAGE sample buffer (50 mM Tris, pH 6.8; 50mM DTT; 2% SDS; 0.2% bromphenol blue; and 10% glycerol). For total cardiac tissue protein extracts, part of the initial homogenate was treated with buffer A, without 0.6 M KCl but with 0.5% SDS added. SDS-PAGE was carried out under standard conditions. Resolved proteins were transferred by electroblotting to polyvinylide difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) and probed with the following anti-desmin antibodies, as indicated in Results: 1) anti-desmin polyclonal antibody (Sigma), 1:200 dilution; 2) anti-desmin N15, 1:30 dilution; and 3) anti-desmin polyclonal antibody H-76 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; raised against desmin amino acids 15–90) at 1:300 dilution. Secondary horseradish peroxidase-conjugated antibodies were from Bio-Rad, and visualization of the peroxidase was performed with enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK). Two-dimensional gel electrophoresis Enriched cytoskeletal protein extracts from cardiac tissue (⬃0.04 mg) were applied on immobilized pH 3–10 nonlinear gradient strips (Bio-Rad), and two-dimensional gel electrophoresis was performed, as previously described (24). 3319
Mass spectroscopy
RESULTS
Tryptic digestion was performed as described previously (24). Samples were analyzed in a time-of-flight (TOF) mass spectrometer (Ultraflex TOF-TOF; Bruker Daltonics, Bremen, Germany) as well as in a nanoelectrospray-source hybrid quadrupole TOF tandem mass spectrometry system (nESIQqTOF MS-MS; QSTAR XL model; Applied Biosystems, Mississauga, ON, Canada) as described previously (25).
A missense mutation in the carboxyl terminus of desmin(I451M) promotes cleavage at the amino terminus
Primary culture of neonatal cardiomyocytes Hearts were harvested from 3-day-old neonatal mice, and after removal of the atria, the ventricles were subjected to trypsin (Life Technologies, Inc., Grand Island, NY, USA) digestion, in a final concentration of 100 g/ml, for 16 –18 h at 4°C. Digestion with collagenase (Life Technologies, Inc.) at a concentration of 150 U/ml followed, and the mixture of cells was plated in collagen-coated chamber slides at a concentration of 105 cells/cm2. The transfection of primary cardiomyocytes with plasmid DNAs was performed using a rat cardiomyocyte-neonatal nucleofector kit (Amaxa Biosystems, Koln, Germany) according to the manufacturer’s instructions. Four days after the transfection, neonatal cardiomyocytes were analyzed by immunofluorescence staining for desmin and ␣-actinin as described previously for cardiac tissue sections. The wild-type (WT) and I451M mouse desmin cDNAs were subcloned into the pRc/CMV plasmid (Invitrogen Corporation, Carlsbad, CA, USA), under the control of CMV promoter. Histology Routine histological analysis and hematoxylin-eosin staining was performed as described previously (2). Paraffin sections (5 m thick) from cardiac tissue, fixed overnight in 2% paraformaldehyde solution in PBS, were used.
To elucidate the role of the missense mutation Ile 451 to Met at the tail domain of the desmin molecule in dilated cardiomyopathy, two transgenic mouse lines, a-TgdesI451M and b-TgdesI451M, were generated. The mutant murine desmin(I451M) cDNA was expressed specifically at the myocardium under the control of the cardiac specific ␣-myosin heavy-chain promoter. Both lines were crossed into the desmin-null (des⫺/⫺) background to generate mice hetero- and homozygous for the mutation. Western blot analysis of cardiac extracts revealed that both lines homozygous for the mutation were expressing desmin(I451M) at lower levels compared to the WT mother lines (Fig. 1). Most importantly, SDS-PAGE revealed that the apparent molecular mass of the mutant desmin was 2– 4 kDa lower than that of the WT (Fig. 1). Western blot analysis of the soluble (3000 g supernatant) and insoluble (0.6 M KCl pellet) fractions, generated from the cardiac tissue IF protein preparation (Fig. 1C), revealed an increase (17.6⫾3.5 vs. 9.6⫾2%, n⫽3) in the desmin(I451M) soluble fraction, relative to WT. The apparent molecular mass of the WT desmin in the SDS-PAGE was calculated as ⬃53–54 kDa, and that of desmin(I451M) as 51–51.5 kDa. The theoretical molecular mass of WT mouse desmin, estimated from the cDNA sequence without any modifications, is 53.497 Da. Differential electrophoretic mobility of desmin(I451M) in SDS-PAGE due to conformational changes was excluded by analyzing the samples after
Figure 1. A mutation at the tail domain of desmin(I451M) expressed in the myocardium of transgenic mice (TgdesI451M) has an apparent molecular mass 2– 4 kDa lower than expected. A) Immunoblot analysis for desmin, performed using cardiac protein extracts from mice WT (lane 1), des⫺/⫺ (lane 2), homozygote des⫺/⫺/aTgdesI451M (lane 3), homozygote des⫺/⫺/bTgdesI451M (lane 4), and heterozygote desWT/bTgdesI451M for the mutation (lane 5). The ␣-desmin polyclonal antibody (Sigma) was used. B) For loading control, same amounts of protein extracts (as calculated by Bradford) used for Western blot were analyzed in the same SDS-PAGE, blotted to PVDF membrane, and stained with Brilliant Blue R (numbering of lanes same as in A). C) Western blot analysis of the soluble fraction (supernatant of 3000 g), insoluble fraction (0.6 M KCl pellet), and total cardiac protein extracts reveals that the major part of the mutant desmin(I451M) is present in the insoluble fraction (numbering of lanes same as in A). 3320
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Figure 2. Mutant desmin(I451M) has an intact carboxyl terminus. Mass spectroscopy was performed on the mutant desmin(I451M) protein, isolated as a band from an SDS-PAGE of the corresponding cardiac extracts. The peptides identified in the mouse desmin sequence are boxed. No peptides were found to cover the first 58 aa sequence of the amino terminus. The mutant amino acid (I/M) is highlighted.
treatment with dithiothreitol and iodoacetamide in the presence of 7 M urea. The 2– 4 kDa reduction in the molecular mass of desmin(Ile451Met) could be explained either by removal of an ⬃20 –30 amino acid (aa) peptide from the carboxyl or amino terminus of the molecule, or by loss of a post-translational modification. To determine the source of the observed difference in the electrophoretic mobility between WT and desmin(I451M), the protein was isolated from the gel and analyzed by tryptic digestion and mass spectrometry. Peptides covering a major part of the protein sequence (⬎60%) were identified (Fig. 2) having no modifications and with an intact carboxyl terminus. No peptides were found covering the first 58 aa of the amino terminus, suggesting a potential removal of part of it. Attempts to sequence the amino terminus of desmin(I451M) by automated Edman degradation were unsuccessful, presumably due to blockage of the amino terminus. To establish that the reduction in molecular mass of desmin(I451M) is due to a cleavage or modification at the very beginning of the amino terminus, we generated an antibody against the first 15 aa of the amino terminus (see Materials and Methods). Analysis of cardiac extracts by Western blot revealed that this antibody was able to recognize the WT 53–54 kDa desmin (Fig. 3A, lane 1) and the WT desmin expressed under the control of aMHC promoter (Fig. 3A, lane 3; the des⫺/⫺/TgdesaMHCdes animals have been previously described in ref. 26), but not the 51 kDa mutant (I451M) form. However, the antibody recognized a small fragment at the front of the SDSPAGE (Fig. 3A, line 4), strongly demonstrating that the N terminus of the mutant desmin(I451M) was specifically cleaved, releasing the ⬃20 –30 aa peptide. Finally, analysis by two-dimensional gel electrophoresis revealed that desmin(I451M) is indeed more acidic than WT desmin (Fig. 4). This could be explained by loss of the first 20 –30 aa peptide of desmin, which is very basic (10.90 theoretical isoelectric point). The estimated isoelectric point for WT desmin is 5.21, whereas for a desmin molecule missing the first 20 aa, it is 5.09. All the above results prompt us to propose that a mutation at the 451 aa position of the C-terminal tail domain of desmin, changing an Ile to Met, promotes a specific cleavage at the N terminus (approximately the first 20 –30 aa) of desmin in cardiomyocytes. The difference in the abundance of the mutant protein between hearts homo- and heterozygous for the mutation (Fig. 1A) suggests that the mutant desmin is less stable and that the presence of WT desmin in the DESMIN ILE451MET MUTATION
heterozygote increases the stability of the mutant. Alternatively, the cardiomyocytes have an internal control mechanism that keeps steady-state levels of desmin protein engaged with cellular structures such as intercalated discs, Z discs, etc., as previously suggested (26). In the case of hearts homozygous for the mutation, the incapability of desmin to interact with Z discs has as a result its degradation. Mutations in desmin, causing aggregate formation, might evade or impair the function of this internal control mechanism (ref. 27 and unpublished results), resulting in increased desmin content in cardiomyocytes. In addition, the mass spectroscopy analysis of the lower molecular weight band from heterozygote extracts revealed the presence of WT C-terminal peptides. This finding shows that the mutation also affects in trans the stability of the N terminus of the WT molecule. Mutant desmin(I451M) forms filaments in primary cardiomyocytes To determine the effect of the I451M mutation on desmin filament formation, primary cardiomyocytes isolated from WT and des⫺/⫺ animals were transfected with
Figure 3. The I451M mutation at the desmin carboxyl terminus promotes cleavage at the amino terminus. Immunoblot analysis of protein extracts from cardiac tissue of WT (lane 1), des⫺/⫺ (lane 2), des⫺/⫺/TgdesaMHCdes (lane 3), and des⫺/⫺/TgdesI451M animals (lane 4), performed using either an antibody (␣-desN15) against the first 15 aa of desmin (A), an antibody (H-76; Santa Cruz) against desmin aa 15–90 (B), or the preimmune serum of ␣-desN15 antibody (C). Note that in A, lane 4, only the cleaved small amino terminus fragment is detected (夝) and in B, lane 4, the remaining part of the desmin molecule is detected. (Sample 4 is overloaded, compared to other samples). 3321
DISCUSSION The present work demonstrates, for the first time, a critical role of an intact desmin head domain in the formation of the IF scaffold around Z discs and also shows a very novel trans interplay between the desmin Figure 4. The mutant (I451M) desmin is more acidic compared to WT desmin. Protein extracts from cardiac tissue of des⫹/–/bTgdesI451M animals, analyzed by two-dimensional gel and probed with anti-desmin polyclonal antibody.
WT or mutant I451M desmin cDNAs, designed to be expressed under the control of CMV promoter (Fig. 5). Immunofluoresence microscopy revealed the presence of normally extended desmin filaments generated from both constructs in both WT and des⫺/⫺ cardiomyocytes. The only difference observed was in des⫺/⫺ cardiomyocytes, where the mutant (I451M) desmin showed higher preference for perinuclear rather than peripheral localization. Analysis by Western blot of protein extracts for desmin, from des⫺/⫺ cardiomyocytes transfected with WT or mutant I451M desmin cDNAs, revealed that both desmin molecules had the same apparent molecular mass as the WT desmin (data not shown). Mutant desmin(I451M) loses its Z-disc localization in cardiomyocytes Immunofluorescence microscopy of cardiac tissue from transgenic animals expressing only the mutant desmin (des⫺/⫺TgdesI451M) demonstrated that, in contrast to WT desmin, the mutant protein loses its ability to associate with the Z disc. However, it retains its association with the intercalated disc (Fig. 6). Intercalated discs in animals expressing only the mutant desmin (des⫺/⫺TgdesI451M) have altered architecture (Fig. 7), especially in older animals. Desmoplakin staining of cardiac tissue sections of transgenic animals (des⫺/⫺TgdesI451M) showed that the intercalated discs have increased width, covering a broader area compared to WT animals (Fig. 7). Desmin-null animals also have altered intercalated discs compared to WT (as has been previously described; refs. 28, 29) but to a lesser extent compared to mutant transgenic animals. In animals expressing only the mutant desmin (des⫺/⫺TgdesI451M), the Z discs are formed properly, as indicated by immunofluorescence staining for sarcomeric ␣-actinin (Fig. 8). Transgenic animals heterozygous for the mutant desmin(I451M) (des⫹/– TgdesI451M) had a similar immunofluorescence staining pattern for desmin as the WT animals (data not shown). Histological observation of cardiac tissue sections from 10-month-old transgenic animals (des⫺/⫺ TgdesI451M), stained by hematoxylin and eosin (Fig. 9) or Mason’s trichrome stain (not shown), revealed a pattern similar to WT without obvious fibrosis or other lesions. Furthermore, immunofluorescence staining of cardiac tissue sections for ubiquitin or ␣〉-crystallin did not reveal any aggregates (data not shown). 3322
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Figure 5. Mutant desmin(I451M) forms filaments in primary cardiomyocytes. Primary cardiomyocytes isolated from WT and des⫺/⫺ animals were transfected with WT desmin or mutant desminI451M cDNAs under the control of CMV promoter and stained for desmin (red) and ␣-actinin (green). In WT cardiomyocytes transfected either with WT (A) or mutant (I451M) desmin (B) and in des⫺/⫺ cardiomyocytes transfected with WT desmin (C), extended filaments formed and are more abundant in the periphery of the cardiomyocytes. In des⫺/⫺ cardiomyocytes transfected with mutant (I451M) desmin (D), abundant desmin staining is observed around the nucleus, as well as in the periphery. In A1, B1, C1, and D1, only the red channel (desmin) is shown for better resolution.
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Figure 6. The mutant (I451M) desmin loses its ability to associate with the Z discs, although its association with the intercalated disc is retained. Immunofluorescence staining for desmin of frozen cardiac tissue sections from 8-month old WT (A) and des⫺/⫺/TgdesI451M mice (B) using the DE-U-10 anti-desmin monoclonal antibody. Image derived after deconvolution processing.
tail and head domains in cardiomyocytes. This interplay could affect the susceptibility of desmin to specific proteases. Whether this head-tail interplay takes place with WT desmin in vivo, or it is a gain of function only due to the specific I451M mutation, remains elusive. Diseases attributed to desmin mutations are fully penetrant, with a spectrum of phenotypes including skeletal myopathy, mixed skeletal-cardiac disease (“desmin-related myopathy”), and cardiomyopathy (dilated and restrictive cardiomyopathy, in most cases accompanied by conduction system disturbance) (8, 30 –32). The prevalence of desmin mutations in dilated cardiomyopathy is 1–2% (33). Two exceptions are reported to date, documenting incomplete penetrance by the age of the expected clinical onset. Both have been observed in families carrying the I451M mutation DESMIN ILE451MET MUTATION
in the desmin tail domain (12, 13). Expression of a patient’s cDNA containing the I451M mutation in cell lines (expressing other IFs or not) led to formation of apparently normal filament network, indicating that the I451M mutation does not prevent normal assembly of desmin filaments (ref. 13 and unpublished results). It has also been shown that under standard in vitro assembly conditions, recombinant desmin I451M is assembled into smooth filaments, which could give rise to extended filamentous arrays with high viscosity (34). By secondary-structure prediction, it has been proposed that in I451M mutant desmin, substitution of isoleucine by methionine removes a turn and replaces a -sheet with an extension of the ␣-helix in the preceding region (12). As such, it has the propensity to be stabilized by interaction with another ␣-helix, including itself (35). This could affect filament thickness, elongation, or other associations. Although many studies have demonstrated filament formation in the absence of intact tail domains, many others have suggested that the tail domain does play a role in assembly. If any consensus can be drawn, it would appear that the tail domain plays a subtle role in filament formation or stability, although it seems to be essential for width control (for review, see refs. 34, 36 and references therein). Moreover, it has been reported that this region binds to the carboxy-terminal part of the rod domain, resulting in the formation of a “loop” or “hairpin” structure that may regulate filament thickness (16). Thus, this conserved -turn motif seems to be essential for interprotofibrillar stability and width control. There are also indications that the head domain may interact with the coil 2B domain, bringing it into proximity with the tail domain. Circular dichroism experiments with recombinant vimentin assembled in vitro have shown that during the elongation of the unit-length filaments (ULFs) to extended filaments, the tyrosines, situated mainly in the head and coil 2B domains, lose their flexibility and become more rigid (S. Georgakopoulou, personal communication). It is also known that during the assembly of protofilaments, the antiparallel mode of arrangement of desmin dimers could bring into proximity the head and tail domains (37). Therefore, alterations in one end could influence, in trans, the opposite end of the closest molecule, thus allowing access of other cellular proteins to these domains and modification of their properties. Filaments formed without a normal tail domain may be more sensitive to external destabilizing forces, which could affect their assembly or protease resistance. No prediction or hypothesis has been reported on factors that could increase the susceptibility of the N terminus to specific proteases. A Ca⫹2-activated proteinase with no absolute sequence specificity, but with high substrate specificity for desmin and vimentin, has been previously reported to attack the ends of filaments and remove most of the head sequence (38). Another study (39) showed that a small tag (myc) at the C terminus of desmin can cause a cleavage of its N terminus (al3323
Figure 7. The demin mutation (I451M) leads to altered desmoplakin (DP) appearance at the intercalated discs of the heart. Intercalated discs in transgenic animals expressing only the mutant desmin (des⫺/⫺TgdesI451M) (B) show increased width of DP-covered area compared to WT animals (A). Desmin-null (des⫺/⫺) animals (C) also have altered intercalated discs compared to WT animals, but to a lesser extent compared to transgenic animals. Frozen cardiac tissue sections of 14-month-old mice were stained for DP. Confocal images of a Z stack of 5 optical sections, 1.5 m total thickness.
though with no specificity), when expressed in neonatal rat cardiac myocytes. However, this does not occur when the additional peptide is much bigger, as is the case when desmin is fused to GFP. In the latter case, the
Figure 8. The Z discs are formed properly in transgenic mice expressing only the mutant (I451M) desmin (des⫺/⫺/ TgdesI451M), as indicated by double immunofluorescence staining with ␣-sarcomeric actinin. Mutant desmin(I451M) is localized only at the intercalated discs. Frozen sections of cardiac tissue from 8-month-old mice were stained with the anti-desmin polyclonal antibody (green) and with anti-␣actinin monoclonal antibody (red). 3324
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fusion protein fails to form filaments in vitro (in contrast to desmin-myc), thus suggesting (in light of our results) that filament formation is required for this effect, potentially because it brings the two termini closer to each other and thus influences accessibility of the N terminus to proteases. The specific cleavage at the N terminus of desmin(I451M), shown by the present work, removes approximately the first 20 –30 aa, which include a conserved peptide motif (SSYRRTFGG). The role of this motif in filament formation was investigated by mutagenesis in Xenopus vimentin (40). Although it was demonstrated not to be essential for IF formation, various mutations within this motif caused severe assembly defects. Furthermore, Kaufmann et al. (41) have shown that desmin, lacking the first 67 aa residues, is incapable of forming structures of a higher order than tetrameric complexes. However, copolymerization of the head-truncated desmin fragment with intact desmin was possible to some degree. Hence, these results indicate that the head domain is required for appropriate dimer– dimer interaction, eventually leading to ULF formation. Structural modifications of the amino and carboxyl termini of IF proteins might exert great influence on the intracellular distribution, molecular organization, association with other molecules, and function of IFs under various physiological and pathological conditions. Indeed, the mutant desmin(I451M) loses its Z-disc localization. Our observation that the mutant desmin(I451M) retains its association with intercalated discs suggests a different mechanism of association of
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Figure 9. The I451M mutation in desmin does not lead to any detectable histological myocardial abnormalities. Hematoxylin-eosin staining of cardiac tissue sections from 10-month-old des⫹/–/TgdesI451M (C) and des⫺/⫺/TgdesI451M animals (D) shows a pattern similar to WT (A), with no obvious fibrosis or other lesions characteristic of the des⫺/⫺ myocardium (B).
desmin to these specialized cell-cell junctions than to Z discs. However, it does not suggest that the intercalated discs are formed and function properly. Indeed, we have shown that the architecture of intercalated discs in transgenic animals expressing the mutant desmin (des⫺/⫺TgdesI451M) is altered and could contribute to the dilated cardiomyopathy observed in affected individuals, more than the lack of desmin from the Z discs. Indeed, analysis of different animal models for dilated cardiomyopathy has suggested that alterations at the intercalated disc structure, namely increased width, as seen under the light microscope, and a higher degree of membrane interdigitation between the neighboring cardiomyocytes, as revealed by electron microscopy, could serve as a hallmark for this disease (42). The present data suggest, but do not prove, that the differential localization of the desmin molecule to specific cellular compartments reflects, in addition to association of different proteins, involvement of different parts of the desmin molecule in these interactions. It is well documented that IFs associate with the desmosomes, and more specifically with the recently defined “area composita” (43), of the intercalated discs through DP (see ref. 42 for review), known to be absent from Z discs. The tail domain of desmin is implicated in the interaction with DP (44). Our assumption is that desmin binds to the Z disc with its head domain, potentially through plectin or ␣-actinin, but this remains elusive. It has been shown that in myocardial sections from patients with a mutation leading to a truncated DP protein (Carvajal syndrome) (45), desmin does not localize at intercalated discs. This suggests that impairment in the interaction of DP with desmin and other proteins might be the underlying causative mechanism. As mentioned above, there are sequences in the desmin tail domain that contribute to DP interaction, though the overall findings do not exclude a critical role of the desmin rod domain for this binding. In yeast 2-hybrid assays, desmin(I451M) DESMIN ILE451MET MUTATION
and desmin with the deleted tail domain abrogate their binding to DP. In vitro binding assays and transfection studies have shown that DP is still able to associate and become coaligned with tailless desmin or desmin(I451M) (44). The researchers suggest that the assembly state of desmin (tetrameric vs. filamentous) may have a critical effect on the conformation and/or the number of available recognition sites important for their interaction with DP. We speculate and propose that alterations in the desmin tail domain play a subtle role in filament formation or stability, but in the context of highly organized muscle cells, these alterations could lead to disturbances of the amino terminus and accessibility to proteases, which have a more severe effect in desmin conformation and its association with the Z discs. Recent data have shown the important role of the desmin amino terminus during embryonic development. Specifically, it was shown that constitutive expression of desmin lacking the first 48 aa residues, in embryonic stem cells, interferes with the beginning of cardiomyogenesis, causing down-regulation of mesodermal and myocardial transcription factors and hampering myofibrillogenesis (46) and survival of cardiomyocytes (47). However, the lack of detectable histological abnormalities in transgenic animals (des⫺/⫺ TgdesI451M) suggests that the loss of desmin localization from the Z discs is not that crucial for the structural integrity of the cardiomyocyte, at least in nonstressed mouse hearts, consistent with our previous observations (48). It also indicates that the time frame in which the alteration of the desmin N terminus occurs is critical for the severity of the phenotype. As suggested from the present data and other studies, in contrast to Z lines, the association of desmin with the intercalated discs, mitochondria, other organelles such as lysosomes (49), and the nucleus might potentially be more important for the function of cardiomyocytes (1). Indeed, the improvement of the desmin-null cardiomyocyte ultrastructure, particu3325
larly the lateral alignment of myofibrils, by overexpression of Bcl-2 (48) has suggested that desmin is not absolutely necessary for the structural integrity of myofibrils. Therefore, it remains to be shown whether, under stress, the I451M cardiomyocytes have mitochondrial or other defects. Furthermore, it remains to be found whether in humans affected by the desmin mutation, the changes in desmin localization are similar to those found in mice. The authors thank Dr. M. Samiotaki (Fleming Research Center, Athens, Greece) and Drs. K. Vougas and A. Vlachou (Biomedical Research Foundation, Academy of Athens, Athens, Greece) for mass spectroscopy analysis; Dr. M. Fountoulakis (Hoffmann-La Roche, Basel, Switzerland) for automated Edman degradation; and Dr. N. Kostomitsopoulos (Biomedical Research Foundation, Academy of Athens) for rabbit immunization. This work was supported by grant SCOR NIH HL-98, and grants EPAN-YB22, PEP-ATT-39, and PENED-01ED371 from the Greek Secretariat of Research and Development to Y.C. This work is dedicated to the memory of K. Karagiaouris.
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