Mutations in desmin's carboxy-terminal "tail" domain severely modify filament and network mechanics.

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-alpha-helical carboxy-terminal domain ("tail"), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin "tail" mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l(p). Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the "tail" mutants (l(p)=1.0-2.0 microm) and the wild-type desmin (l(p)=1.1+/-0.5 microm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered. In order to explore the importance of the "tail" domain as such for filament network properties, we employed a "tail"-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the "tail" domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.

Disease mutations in the "head" domain of the extra-sarcomeric protein desmin distinctly alter its assembly and network-forming properties.

The intermediate filament protein desmin generates an extra-sarcomeric network in myocytes. Mutations in the desmin gene cause myofibrillar myopathy characterized by desmin-positive aggregates and myofibrillar dissolution. Past analysis revealed that the non-alpha-helical amino-terminal "head" domain of desmin is a vital coordinator of protein assembly. We have now characterized assembly and network-forming properties of five recently discovered myopathy-causing mutations residing in this domain. In vitro analyses with recombinant proteins show that two mutant variants residing in a conserved nonapeptide motif "SSYRRTFGG"-Ser13Phe and Arg16Cys-interfere with assembly by forming filamentous aggregates. Consistent with in vitro data, both mutant proteins are unable to generate a bona fide filament system in cells lacking an intermediate filament cytoskeleton. In cells expressing vimentin or desmin, both mutants firstly fail to integrate into the endogenous filament network and secondly severely affect its cellular localization. The other three mutations-Ser2Iso, Ser46Phe, and Ser46Tyr-influence in vitro filament properties less severely, but in vivo, Ser46Phe and Ser46Tyr impair de novo filament formation. These effects of the "head" mutant proteins on endogenous intermediate filament system and their competition for binding to cellular anchoring structures might explain part of the molecular mechanism that causes disease.

Interference of amino-terminal desmin fragments with desmin filament formation.

Short polypeptides from intermediate filament (IF) proteins containing one of the two IF-consensus motifs interfere severely with filament assembly in vitro. We now have systematically investigated a series of larger fragments of the muscle-specific IF protein desmin representing entire functional domains such as coil1 or coil 2. "Half molecules" comprising the amino-terminal portion of desmin, such as DesDeltaC240 and the "tagged" derivative Des(ESA)DeltaC244, assembled into large, roundish aggregates already at low ionic strength, DesDeltaC250 formed extended, relatively uniform filaments, whereas DesDeltaC265 and DesDeltaC300 were soluble under these conditions. Surprisingly, all mutant desmin fragments assembled very rapidly into long thick filaments or spacious aggregates when the ionic strength was raised to standard assembly conditions. In contrast, when these desmin mutants were assembled in the presence of wild-type (WT) desmin, their assembly properties were completely changed: The elongation of the two shorter desmin fragments was completely inhibited by WT desmin, whereas DesDeltaC250, DesDeltaC265 and DesDeltaC300 coassembled with desmin into filaments, but these mixed filaments were distinctly disturbed and exhibited a very different phenotype for each mutant. After transfection into fibroblasts and cardiomyocytes, the truncated mutant Des (ESA)DeltaC244 localized largely to the cytoplasm, as revealed by a tag-specific monoclonal antibody, and also partially colocalized there with the collapsed endogenous vimentin and desmin systems indicating its interference with IF-organizing processes. In contrast, in cells without an authentic cytoplasmic IF system such as line SW13, Des(ESA)DeltaC242 entered the nucleus and was deposited in small dot-like structures in chromatin-free spaces without any noticeable effect on nuclear morphology.

Desmin and vimentin intermediate filament networks: their viscoelastic properties investigated by mechanical rheometry.

We have investigated the viscoelastic properties of the cytoplasmic intermediate filament (IF) proteins desmin and vimentin. Mechanical measurements were supported by time-dependent electron microscopy studies of the assembly process under similar conditions. Network formation starts within 2 min, but it takes more than 30 min until equilibrium mechanical network strength is reached. Filament bundling is more pronounced for desmin than for vimentin. Desmin filaments (persistence length l(p) approximately 900 nm) are stiffer than vimentin filaments (l(p) approximately 400 nm), but both IFs are much more flexible than microfilaments. The concentration dependence of the plateau modulus G(0) approximately c(alpha) is much weaker than predicted theoretically for networks of semiflexible filaments. This is more pronounced for vimentin (alpha=0.47) than for desmin (alpha=0.70). Both networks exhibit strain stiffening at large shear deformations. At the transition from linear to nonlinear viscoelastic response, only desmin shows characteristics of nonaffine network deformation. Strain stiffening and the maximum modulus occur at strain amplitudes about an order of magnitude larger than those for microfilaments. This is probably attributable to axial slippage within the tetramer building blocks of the IFs. Network deformation beyond a critical strain gamma(max) results in irreversible damage. Strain stiffening sets in at lower concentrations, is more pronounced, and is less sensitive to ionic strength for desmin than for vimentin. Hence, desmin exhibits strain stiffening even at low-salt concentrations, which is not observed for vimentin, and we conclude that the strength of electrostatic repulsion compared to the strength of attractive interactions forming the network junctions is significantly weaker for desmin than for vimentin filaments. These findings indicate that both IFs exhibit distinct mechanical properties that are adapted to their respective cellular surroundings [i.e., myocytes (desmin) and fibroblasts (vimentin)].

Prevalence of asymptomatic mitral valve malformations.

INTRODUCTION: Congenital abnormalities of the mitral valve are considered to be very rare cardiac anomalies. In particular, more severe malformations, such as the complete absence of either aortic (anterior) or mural (posterior) mitral leaflet, are usually considered to be incompatible with life. Ebstein-like malformation of the mitral valve is an extremely rare form of mitral valve deformity hitherto unreported in an asymptomatic adult patient. MATERIALS AND METHODS: The detection of such a malformation prompted us to perform a prospective analysis of 26,484 consecutive comprehensive 2D-echocardiographic examinations, conducted at our tertiary care university hospital between April 2007 and July 2008, with regard to the presence of malformations of the mitral valvular apparatus. RESULTS: In total, we found three cases of hypoplastic or even absent functional mural valve leaflets. All were diagnosed in adult patients who attended our outpatient department and were surprisingly asymptomatic regarding this finding. From our patient cohort, we calculate an actual prevalence of asymptomatic hypoplasia of the mitral valve of 1:8,800. CONCLUSIONS: Our findings broaden the spectrum of known mitral valve pathologies. The comparatively high prevalence of this malformation in our preselected patient cohort might indicate that this particular malformation has so far been under-diagnosed. In the context of this observation, both embryological development of the atrioventricular (AV) valves and recent functional insights into mitral valve physiology gained by mitral valve reconstructive surgery are discussed.

Accessory mitral valve as a potential source of cardioembolism.

An accessory mitral valve (AMV) is considered to arise from abnormal development of endocardial cushion tissue. It is a very rare entity, commonly diagnosed in childhood and associated with symptomatic left ventricular outflow tract (LVOT) obstruction. Here we describe the presence of AMV in a 58-year old patient who presented with a transient ischemic attack. Transesophageal echocardiography visualized a spherical structure attached to the ventricular aspect of the anterior mitral valve leaflet.

Biventricular involvement in transient apical ballooning syndrome.

Transient apical ballooning syndrome (Takotsubo cardiomyopathy) is an acute cardiac syndrome mimicking ST-elevation myocardial infarction. It is characterized by ventricular wall motion abnormalities confined to the apical regions of the left ventricle. Here we describe an 80-year old woman presenting with acute shortness of breath. Echocardiography demonstrated left and right ventricular apical akinesia and basal hyperkinesia. Cardiac catheterisation disclosed minimal atherosclerotic changes of the coronary arteries. Both symptoms and echocardiographic findings resolved completely within one week.

Unexpected pulsatile intracardiac mass.

We present a curious pulsatile right atrial mass which we found by routine transthoracic echocardiography in an asymptomatic woman. Only consecutive section planes finally revealed the true nature of this round mass, highlighting the general statement that the ultrasonographer should never rely on a single ultrasound imaging plane for making a correct diagnosis.

Cardiomyocyte production in mass suspension culture: embryonic stem cells as a source for great amounts of functional cardiomyocytes.

Exploiting embryonic stem cell (ESC)-derived cardiomyocytes as a vital source for cell therapies and tissue engineering will depend on robust, large-scale production processes. Recently, we have reported stirring-controlled formation of embryoid bodies, enabling the generation of pure cardiomyocytes in 2-L scale. Expansion and differentiation of genetically engineered mouse ESCs was followed by antibiotic-based cardiomyocyte enrichment. Here we have investigated modification of various parameters aiming at process optimization in a 250-mL spinner flask system. Duration of the differentiation phase, timing of retinoic acid addition, and a slower medium exchange rate were found to be crucial to enhancing cardiomyocyte yield. Improved process conditions were consequently transferred to a 2-L controlled bioreactor. Employing a manual fill-and-draw medium change resulted in the formation of 0.86 x 10(9) cardiomyocytes in a single 2-L batch, thereby reproducing our previous findings. In contrast, an automated perfusion-based strategy enabled the production of 4.6 x 10(9) cardiomyocytes in a single run. This is significantly higher than previously reported and highlights the enormous process optimization potential in the scalable generation of ESC-derived cell lineages.

Clinical, genetic, and cardiac magnetic resonance imaging findings in primary desminopathies.

We report the clinical, genetic and cardiac magnetic resonance imaging (MRI) findings in 11 German patients with heterozygous E245D, D339Y, R350P and L377P desmin mutations and without cardiac symptoms. Clinical evaluation revealed a marked variability of skeletal muscle, respiratory and cardiac involvement even between patients with identical mutations, ranging from asymptomatic to severely affected. While echocardiography did not show any pathological findings in all 11 patients, cine MRI revealed focal left ventricular hypertrophy in 2 patients and MR delayed enhancement imaging displayed intramyocardial fibrosis in the left ventricle in 4 patients indicating early myocardial involvement. Our data argue against distinct genotype-phenotype correlations and suggest that comprehensive cardiac MRI is superior to conventional echocardiography for the detection of early and clinically asymptomatic stages of cardiomyopathy in desminopathy patients. Therefore, cardiac MRI may serve as a screening tool to identify patients at risk, which might benefit from early pharmacological and/or interventional (e.g. implantable cardioverter-defibrillator devices) therapy.

Incomplete nonsense-mediated decay of mutant lamin A/C mRNA provokes dilated cardiomyopathy and ventricular tachycardia.

We have identified a family in which several members died of sudden cardiac death or suffer from dilated cardiomyopathy (DCM) and rhythm disturbances. Mutation screening revealed co-segregation of a novel nonsense mutation (pR321X) in the lamin A gene, LMNA, with the disease. Lamin A, and its smaller splice form lamin C are nuclear intermediate filament proteins forming a major part of the lamina, which is underlying the inner nuclear membrane. They are involved in the organization of heterochromatin and both in DNA replication and transcription. Recently, an increasing number of missense mutations in LMNA have been discovered to cause various types of rare diseases. Here, we investigated the causal role of the new nonsense mutation for the disease. Quantification of wild type and mutant lamin A mRNA from explanted myocardial tissue and cultured fibroblasts revealed an up to 30-fold reduction in the relative amount of the mutant transcript indicating that its synthesis was massively down-regulated by nonsense-mediated mRNA decay (NMD). Correspondingly, we did not detect the mutant truncated lamin A by Western blot analysis in extracts of patient fibroblasts and cardiac muscle tissue. Both wild type lamin A and C were present, however, in normal quantities. The immunohistochemical analyses of patient tissues revealed a normal distribution of lamin A/C and of major inner nuclear membrane proteins such as emerin and the lamin B receptor. Moreover, both chromatin distribution and nuclear shape were normal. However, over-expression of truncated lamin A in HeLa cells by transient transfection caused major disturbances of lamin A organization within both the nucleoplasm and the cytoplasm. In addition, after treatment of patient fibroblasts with the proteasome inhibitor epoxomicin, mutant truncated lamin A was detected in relatively high levels by Western blotting demonstrating that it is synthesized in these cells. Therefore, we conclude that NMD is not sufficient to completely prevent the expression of truncated lamin A and that even trace amounts of it may negatively interfere with structural and/or regulatory functions of lamin A/C eventually leading to the development of DCM and rhythm disturbances.

Intermediate filaments: from cell architecture to nanomechanics.

Intermediate filaments (IFs) constitute a major structural element of animal cells. They build two distinct systems, one in the nucleus and one in the cytoplasm. In both cases, their major function is assumed to be that of a mechanical stress absorber and an integrating device for the entire cytoskeleton. In line with this, recent disease mutations in human IF proteins indicate that the nanomechanical properties of cell-type-specific IFs are central to the pathogenesis of diseases as diverse as muscular dystrophy and premature ageing. However, the analysis of these various diseases suggests that IFs also have an important role in cell-type-specific physiological functions.

Conspicuous involvement of desmin tail mutations in diverse cardiac and skeletal myopathies.

Myofibrillar myopathy (MFM) encompasses a genetically heterogeneous group of human diseases caused by mutations in genes coding for structural proteins of muscle. Mutations in the intermediate filament (IF) protein desmin (DES), a major cytoskeletal component of myocytes, lead to severe forms of "desminopathy," which affects cardiac, skeletal, and smooth muscle. Most mutations described reside in the central alpha-helical rod domain of desmin. Here we report three novel mutations--c.1325C>T (p.T442I), c.1360C>T (p.R454W), and c.1379G>T (p.S460I)--located in desmin's non-alpha-helical carboxy-terminal "tail" domain. We have investigated the impact of these and four--c.1237G>A (p.E413K), c.1346A>C (p.K449T), c.1353C>G (p.I451M), and c.1405G>A (p.V469M)--previously described "tail" mutations on in vitro filament formation and on the generation of ordered cytoskeletal arrays in transfected myoblasts. Although all but two mutants (p.E413K, p.R454W) assembled into IFs in vitro and all except p.E413K were incorporated into IF arrays in transfected C2C12 cells, filament properties differed significantly from wild-type desmin as revealed by viscometric assembly assays. Most notably, when coassembled with wild-type desmin, these mutants revealed a severe disturbance of filament-formation competence and filament-filament interactions, indicating an inherent incompatibility of mutant and wild-type protein to form mixed filaments. The various clinical phenotypes observed may reflect altered interactions of desmin's tail domain with different components of the myoblast cytoskeleton leading to diminished biomechanical properties and/or altered metabolism of the individual myocyte. Our in vitro assembly regimen proved to be a very sensible tool to detect if a particular desmin mutation is able to cause filament abnormalities.

Assembly defects of desmin disease mutants carrying deletions in the alpha-helical rod domain are rescued by wild type protein.

Most mutations of desmin that cause severe autosomal dominant forms of myofibrillar myopathy are point mutations and locate in the central alpha-helical coiled-coil rod domain. Recently, two in-frame deletions of one and three amino acids, respectively, in the alpha-helix have been described and discussed to drastically interfere with the architecture of the desmin dimer and possibly also the formation of tetramers and higher order complexes [Kaminska, A., Strelkov, S.V., Goudeau, B., Olive, M., Dagvadorj, A., Fidzianska, A., Simon-Casteras, M., Shatunov, A., Dalakas, M.C., Ferrer, I., Kwiecinski, H., Vicart, P., Goldfarb, L.G., 2004. Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy. Hum. Genet. 114, 306-313.]. Therefore, it was proposed that they may poison intermediate filament (IF) assembly. We have now recombinantly synthesized both mutant proteins and subjected them to comprehensive in vitro assembly experiments. While exhibiting assembly defects when analyzed on their own, both one-to-one mixtures of the respective mutant protein with wild type desmin facilitated proper filament formation. Transient transfection studies complemented this fundamental finding by demonstrating that wild type desmin is also rescuing these assembly defects in vivo. In summary, our findings strongly question the previous hypothesis that it is assembly incompetence due to molecular rearrangements caused by the mutations, which triggers the development of disease. As an alternative, we propose that these mutations cause subtle age-dependent structural alterations of desmin IFs that eventually lead to disease.

Impact of disease mutations on the desmin filament assembly process.

It has been documented that mutations in the human desmin gene lead to a severe type of myofibrillar myopathy, termed more specifically desminopathy, which affects cardiac and skeletal as well as smooth muscle. We showed recently that 14 recombinant versions of these disease-causing desmin variants, all involving single amino acid substitutions in the alpha-helical rod domain, interfere with in vitro filament formation at distinct stages of the assembly process. We now provide mechanistic details of how these mutations affect the filament assembly process by employing analytical ultracentrifugation, time-lapse electron microscopy of negatively stained and glycerol-sprayed/low-angle rotary metal-shadowed samples, quantitative scanning transmission electron microscopy, and viscometric studies. In particular, the soluble assembly intermediates of two of the mutated proteins exhibit unusually high s-values, compatible with octamers and other higher-order complexes. Moreover, several of the six filament-forming mutant variants deviated considerably from wild-type desmin with respect to their filament diameters and mass-per-length values. In the heteropolymeric situation with wild-type desmin, four of the mutant variants caused a pronounced "hyper-assembly", when assayed by viscometry. This indicates that the various mutations may cause abortion of filament formation by the mutant protein at distinct stages, and that some of them interfere severely with the assembly of wild-type desmin. Taken together, our findings provide novel insights into the basic intermediate filament assembly mechanisms and offer clues as to how amino acid changes within the desmin rod domain may interfere with the normal structural organization of the muscle cytoskeleton, eventually leading to desminopathy.

Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation.

We recently demonstrated that inherited disease-causing mutations clustered in the alpha-helical coiled-coil "rod" domain of the muscle-specific intermediate filament (IF) protein desmin display a wide range of inhibitory effects on regular in vitro assembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area.

Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages.

Desmin is the major intermediate filament (IF) protein of muscle. Recently, mutations of the desmin gene have been reported to cause familial or sporadic forms of human skeletal, as well as cardiac, myopathy, termed desmin-related myopathy (DRM). The impact of any of these mutations on filament assembly and integration into the cytoskeletal network of myocytes is currently not understood, despite the fact that all cause the same histopathological defect, i.e., desmin aggregation. To gain more insight into the molecular basis of this process, we investigated how mutations within the alpha-helical rod domain of desmin affect both the assembly of the recombinant protein in vitro as well as the filament-forming capacity in cDNA-transfected cells. Whereas 6 of 14 mutants assemble into seemingly normal IFs in the test tube, the other mutants interfere with the assembly process at distinct stages, i.e., tetramer formation, unit-length filament (ULF) formation, filament elongation, and IF maturation. Correspondingly, the mutants with in vitro assembly defects yield dot-like aggregates in transfected cells, whereas the mutants that form IFs constitute a seemingly normal IF cytoskeleton in the cellular context. At present, it is entirely unclear why the latter mutant proteins also lead to aggregate formation in myocytes. Hence, these findings may be a starting point to dissect the contribution of the individual subdomains for desmin pathology and, eventually, the development of therapeutic interventions.

Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro.

Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Here, we report the pathogenic effects of a novel heterozygous R350P desmin missense mutation, which resides in the evolutionary highly conserved coil 2B domain of the alpha-helical coiled-coil desmin rod domain, on the assembly of desmin intermediate filaments (IF) in cultured cells and in vitro. By transfection experiments, we show that R350P desmin is incapable of de novo formation of a desmin IF network in vimentin-free BMGE+H, MCF7 and SW13 cells and that it disrupts the endogenous vimentin cytoskeleton in 3T3 fibroblast cells. Hence, transfected cells displayed abnormal cytoplasmic protein aggregates reminiscent of desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of the affected family. To study the functional effects of the R350P desmin mutation at the protein level, we performed in vitro assembly studies with wild-type (WT) and mutant desmin protein. Our analysis revealed that the in vitro assembly process of R350P desmin is already disturbed at the unit length filament level and that further association reactions generate huge, tightly packed protein aggregates. On assessing the pathogenic effects of R350P desmin in various mixtures with WT desmin, we show that a ratio of 1 : 3 (R350P desmin/WT desmin) is sufficient to effectively block the normal polymerization process of desmin IFs. Our findings indicate that the heterozygous R350P desmin mutation exerts a dominant negative effect on the ordered lateral arrangement of desmin subunits. This disturbance of the lateral packing taking place in the first phase of assembly is ultimately leading to abnormal protein aggregation.

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.