Biomedical subjects

Elisabeth Ehler

Publications and source records attributed to Elisabeth Ehler.

At least 19 recordsLinked to original sources

PubMed2006

Probing the role of septins in cardiomyocytes.

Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.

Actinin↗

PubMed2006

Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP.

Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z-discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly.

Alternative Splicing↗

PubMed2006

Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats.

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.

Animals↗

PubMed2006

Estrogen receptor alpha up-regulation and redistribution in human heart failure.

Clinical and animal studies suggest that estrogen receptors are involved in the development of myocardial hypertrophy and heart failure. In this study, we investigated whether human myocardial estrogen receptor alpha (ERalpha) expression, localization, and association with structural proteins was altered in end stage-failing hearts. We found a 1.8-fold increase in ERalpha mRNA and protein in end-stage human dilated cardiomyopathy (DCM, n=41), as compared with controls (n=25). ERalpha was visualized by confocal immunofluorescence microscopy and localized to the cytoplasm, sarcolemma, intercalated discs and nuclei of cardiomyocytes. Immunofluorescence studies demonstrated colocalization of ERalpha with beta-catenin at the intercalated disc in control hearts and immunoprecipitation studies confirmed complex formation of both proteins. Interestingly, the ERalpha/beta-catenin colocalization was lost at the intercalated disc in DCM hearts. Thus, the ERalpha/beta-catenin colocalization in the intercalated disc may be of functional relevance and a loss of this association may play a role in the progression of heart failure. The increase of total ERalpha expression may represent a compensatory process to contribute to the stability of cardiac intercalated discs.

Adult↗

PubMed2006

Increased field resistance to Tilletia caries provided by a specific antifungal virus gene in genetically engineered wheat.

The field performance of a viral gene in two Swiss wheat (Triticum aestivum) varieties showed 10% increased fungal resistance against Tilletia caries (stinking smut). To the best of our knowledge, this is the first report of improved resistance against any fungus in the field achieved by genetic engineering in wheat. The genetically modified wheat lines previously showed a c. 30% decrease in symptoms of T. caries in the glasshouse (Clausen, M., Kräuter, R., Schachermayr, G., Potrykus, I. and Sautter, C. (2000) Antifungal activity of a virally encoded gene in transgenic wheat. Nat. Biotechnol. 18, 446-449), depending on the fungal strain inoculated. A glasshouse experiment run in parallel to the field test, and using the same collection of T. caries, gave the same results. In a dose-response experiment with isolated fungal strains, in which the infection pressure was varied via the spore concentration, the transgene behaved as a quantitative resistance gene and shifted the S-shaped dose-response curve towards higher resistance. The transgene was shown to be highly specific for fungi of the order Ustilaginales. Tests of the transgene using cell cultures of eukaryotes, including hamster and human, showed no significant side-effects with respect to biosafety. Endogenous pathogen-related genes were also activated on fungal infection in the presence of the kp4 transgene.

Animals↗

PubMed2005

Establishment of cardiac cytoarchitecture in the developing mouse heart.

Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.

Adherens Junctions↗

PubMed2005

From A to Z and back? Multicompartment proteins in the sarcomere.

Sarcomeres, the smallest contractile units of striated muscle, are conventionally perceived as the most regular macromolecular assemblies in biology, with precisely assigned localizations for their constituent proteins. However, recent studies have revealed complex multiple locations for several sarcomere proteins within the sarcomere and other cellular compartments such as the nucleus. Several of these proteins appear to relocalize in response to mechanical stimuli. Here, we review the emerging role of these protein networks as dynamic information switchboards that communicate between the contractile machinery and the nucleus to central pathways controlling cell survival, protein breakdown, gene expression and extracellular signaling.

Animals↗

PubMed2005

Involvement of palladin and alpha-actinin in targeting of the Abl/Arg kinase adaptor ArgBP2 to the actin cytoskeleton.

Palladin and alpha-actinin are major components of stress fiber dense bodies, cardiomyocyte Z-discs and neuronal synapses. They function as structural molecules and cytoskeletal regulators but also as docking sites to other proteins. Both antisense and transient overexpression experiments have shown that palladin plays an important role in the regulation of actin cytoskeleton. ArgBP2 is a multi-domain scaffolding protein which shares both the tissue distribution and subcellular localization with palladin. ArgBP2 is directly linked to intracellular signaling cascades by its interaction with Abl family kinases, Pyk2 and the ubiquitin ligase Cbl. It has several actin associated binding partners and has been shown to regulate cytoskeletal dynamics. Here, we show by in vivo and in vitro methods that palladin's amino-terminal poly-proline sequences directly interact with the first carboxy-terminal SH3 domain of ArgBP2. We further demonstrate a direct interaction between alpha-actinin and the amino-terminal segment of ArgBP2. Immunoprecipitation and targeting assays suggest that a three-way complex of the proteins occurs in vivo. The interactions provide an explanation to the previously observed Z-disc-specific localization of ArgBP2 and indicate interplay between signaling adaptors and structural proteins of the Z-disc.

Actinin↗

PubMed2005

VEGF profiling and angiogenesis in human microtissues.

Owing to its dual impact on tissue engineering (neovascularization of tissue implants) and cancer treatment (prevention of tumor-induced vascularization), management and elucidation of vascularization phenomena remain clinical priorities. Using a variety of primary human cells and (neoplastic) cell lines assembled in microtissues by gravity-enforced self-aggregation in hanging drops we (i) studied size and age-dependent VEGF production of microtissues in comparison to isogenic monolayer cultures, (ii) characterized the self-organization and VEGF-production potential of mixed-cell spheroids, (iii) analyzed VEGF-dependent capillary formation of human umbilical vein endothelial cells (HUVECs) cells coated onto several human primary cell spheroids, and (iv) profiled endostatin action on vascularization in human microtissues. Precise understanding of vascularization in human microtissues may foster advances in clinical tissue implant engineering, tumor treatment, as well as drug discovery and drug-function analysis.

Aorta↗

PubMed2005

The kinase domain of titin controls muscle gene expression and protein turnover.

The giant sarcomeric protein titin contains a protein kinase domain (TK) ideally positioned to sense mechanical load. We identified a signaling complex where TK interacts with the zinc-finger protein nbr1 through a mechanically inducible conformation. Nbr1 targets the ubiquitin-associated p62/SQSTM1 to sarcomeres, and p62 in turn interacts with MuRF2, a muscle-specific RING-B-box E3 ligase and ligand of the transactivation domain of the serum response transcription factor (SRF). Nuclear translocation of MuRF2 was induced by mechanical inactivity and caused reduction of nuclear SRF and repression of transcription. A human mutation in the titin protein kinase domain causes hereditary muscle disease by disrupting this pathway.

Amino Acid Sequence↗

PubMed2005

Dimerisation of myomesin: implications for the structure of the sarcomeric M-band.

The sarcomeric M-band is thought to provide a link between the thick and the elastic filament systems. So far, relatively little is known about its structural components and their three-dimensional organisation. Myomesin seems to be an essential component of the M-band, since it is expressed in all types of vertebrate striated muscle fibres investigated and can be found in its mature localisation pattern as soon as the first myofibrils are assembled. Previous work has shown that the N-terminal and central part of myomesin harbour binding sites for myosin, titin and muscle creatine kinase. Intrigued by the highly conserved domain layout of the C-terminal half, we screened for new interaction partners by yeast two-hybrid analysis. This revealed a strong interaction of myomesin with itself. This finding was confirmed by several biochemical assays. Our data suggest that myomesin can form antiparallel dimers via a binding site residing in its C-terminal domain 13. We suggest that, similar to alpha-actinin in the Z-disc, the myomesin dimers cross-link the contractile filaments in the M-band. The new and the already previously identified myomesin interaction sites are integrated into the first three-dimensional model of the sarcomeric M-band on a molecular basis.

Animals↗

PubMed2005

The sarcomeric M-band during development and in disease.

The C-terminus of connectin/titin at the M-band of the sarcomere interacts with several structural as well as potential signalling proteins. One of these is myomesin, which can also bind to myosin and has been suggested to function as an integral structural linker of the thick filaments into the sarcomere. Recent evidence that myomesin possesses the ability to form antiparallel dimers via its C-terminal domain has prompted us to propose a novel three-dimensional model for the sarcomeric M-band. A splice variant of myomesin, termed EH-myomesin, contains an additional segment that has disordered conformation and functions as an entropic spring. It is expressed in a subset of muscle types that are characterised by a broader operational range and are more resistant to damage caused by eccentric contraction. In addition, it is also re-expressed in dilated cardiomyopathy. DRAL/FHL-2 is another protein that interacts with the M-band portion of connectin/titin and which probably functions as an adaptor for the compartmentalisation of metabolic enzymes. Together these results suggest that the M-band is crucial for sarcomere function and maintenance and that its molecular composition can be adapted to divergent physiological needs in different muscle types, which may help to cope with pathological alterations.

Animals↗

PubMed2004

Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes.

The contractile tissue of the heart is composed of individual cardiomyocytes. During mammalian embryonic development, heart growth is achieved by cell division while at the same time the heart is already exerting its essential pumping activity. There is still some debate whether the proliferative activity is carried out by a less differentiated, stem cell-like type of cardiomyocytes or whether embryonic cardiomyocytes are able to perform both of these completely different dynamic tasks, contraction and cell division. Our analysis of triple-stained specimen of cultured embryonic cardiomyocytes and of whole mount preparations of embryonic mouse hearts by confocal microscopy revealed that differentiated cardiomyocytes are indeed able to proliferate. However, to go through cell division, a disassembly of the contractile elements, the myofibrils, has to take place. This disassembly occurs in two steps with Z-disk and thin (actin)-filament-associated proteins getting disassembled before disassembly of the M-bands and the thick (myosin) filaments happens. After cytokinesis reassembly of the myofibrillar proteins to their mature cross-striated pattern can be seen. Another interesting observation was that the cell-cell contacts remain seemingly intact during division, probably reflecting the requirement of intact integration sites of the individual cells in the contractile tissue. Our results suggest that embryonic cardiomyocytes have developed an interesting strategy to deal with their major cytoskeletal elements, the myofibrils, during mitosis. The complex disassembly-reassembly process might also provide a mechanistic explanation, why cardiomyocytes cede to divide postnatally.

Actinin↗

PubMed2004

Myofibrillogenesis in the developing chicken heart: role of actin isoforms and of the pointed end actin capping protein tropomodulin during thin filament assembly.

Recently, important differences between myofibrillogenesis in cultured cardiomyocytes vs. the three-dimensional setting in situ could be determined. We investigated thin filament assembly in situ by confocal microscopy of whole-mount preparations of immunostained embryonic chicken hearts. Of interest, a distinct localisation of different actin isoforms was observed in immature thin filaments. Cardiac alpha-actin is restricted to filaments with a length comparable to mature thin filaments as soon as the first contractions occur, while vascular alpha-actin makes up filaments that extend toward the M-band. The pointed-end actin filament capping protein tropomodulin can be found initially in close association with the plasma membrane, but attains its mature localisation pattern at the ends of the thin filaments only comparatively late during myofibrillogenesis. Thus tropomodulin acts as a length stabilising element of actin filaments also in developing cardiomyocytes in situ, but plays an additional role together with membrane-associated actin filaments in the earliest steps of myofibril assembly.

Actin Cytoskeleton↗

PubMed2004

The sensitive giant: the role of titin-based stretch sensing complexes in the heart.

Every heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.

Animals↗

PubMed2004

The molecular composition of the sarcomeric M-band correlates with muscle fiber type.

The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.

Animals↗

PubMed2003

Intermediate filament-co-localized molecules with myosin heavy chain epitopes define distinct cellular domains in hair follicles and epidermis.

BACKGROUND: Proteins linking intermediate filaments to other cytoskeletal components have important functions in maintaining tissue integrity and cell shape. RESULTS: We found a set of monoclonal antibodies raised against specific human sarcomeric myosin heavy chain (MyHC) isoforms labels cells in distinct regions of the mammalian epidermis. The antigens co-localize with intermediate filament-containing structures. A slow MyHC-related antigen is punctate on the cell surface and co-localizes with desmoplakin at desmosomal junctions of all suprabasal epidermal layers from rat foetal day 16 onwards, in the root sheath of the hair follicle and in intercalated disks of cardiomyocytes. A fast MyHC-related antigen occurs in cytoplasmic filaments in a subset of basal cells of skin epidermis and bulb, but not neck, of hair follicles. A fast IIA MyHC-related antigen labels filaments of a single layer of cells in hair bulb. This 230,000 Mr antigen co-purifies with keratin. No obvious candidate for any of the antigens appears in the literature. CONCLUSIONS: We describe a set of molecules that co-localize with intermediate filament in specific cell subsets in epithelial tissues. These antigens presumably influence intermediate filament structure or function.

Animals↗

PubMed2003

Identification, characterization, and expression of a novel alpha-tropomyosin isoform in cardiac tissues in developing chicken.

Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non-muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM-4 type. This novel alpha-Tm tropomyosin isoform, designated as alpha-Tm-2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (alpha-Tm-1) was also expressed in embryonic hearts along with the striated muscle isoform of TM-4. In adult heart, TM-4 was expressed, however, expression of both alpha-Tm-1 and alpha-Tm-2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of alpha-Tm-2 in embryonic and adult skeletal muscle, however, the alpha-Tm-1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the alpha-TM gene, i.e., alpha-smooth muscle tropomyosin (alpha-Sm), alpha-Fibroblast-1 (alpha-F1), and alpha-Fibroblast-2 (alpha-F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of alpha-Tm-1 and alpha-Tm-2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the alpha-TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.

Amino Acid Sequence↗