Biomedical subjects

Elisabeth Ehler

Publications and source records attributed to Elisabeth Ehler.

29 records · Page 2Linked to original sources

PubMed2003

PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions.

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.

3T3 Cells↗

PubMed2003

Dilated cardiomyopathy: a disease of the intercalated disc?

The contractile tissue of the heart is composed of individual cells, making specific cell-cell contacts necessary to ensure mechanical and electrochemical coupling during beating. These contact sites, termed the intercalated discs, have gained increased attention recently due to their potential involvement in cardiac disease. This article discusses how the intercalated discs are assembled during heart development and how they are affected in cardiomyopathy, with particular emphasis on dilated cardiomyopathy. A model is proposed to relate the alterations that are seen at a molecular level with changes in function observed in that kind of cardiac disease.

Animals↗

PubMed2003

M-band: a safeguard for sarcomere stability?

The sarcomere of striated muscle is a very efficient machine transforming chemical energy into movement. However, a wrong distribution of the generated forces may lead to self-destruction of the engine itself. A well-known example for this is eccentric contraction (elongation of the sarcomere in the activated state), which damages sarcomeric structure and leads to a reduced muscle performance. The goal of this review is to discuss the involvement of different cytoskeletal systems, in particular the M-band filaments, in the mechanisms that provide stability during sarcomeric contraction. The M-band is the transverse structure in the center of the sarcomeric A-band, which is responsible both for the regular packing of thick filaments and for the uniform distribution of the tension over the myosin filament lattice in the activated sarcomere. Although some proteins from the Ig-superfamily, like myomesin and M-protein, are the major candidates for the role of M-band bridges, the exact molecular organisation of the M-band is not clear. However, the protein composition of the M-band seems to modulate the mechanical characteristics of the thick filament lattice, in particular its stiffness, adjusting it to the specific demands in different muscle types. The special M-band design in slow fibers might be part of structural adaptations, favouring sarcomere stability for a continuous contractile activity over a broad working range. In conclusion, we discuss why the interference with M-band structure might have fatal consequences for the integrity of the working sarcomere.

Animals↗

PubMed2002

Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2.

During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.

Adenylate Kinase↗

PubMed2002

Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure.

During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O(2) in serum- and glucose-free medium) for 5 h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30 min, strongly inhibited after 5 h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.

Blotting, Western↗

PubMed2002

WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons.

Sensory axons from dorsal root ganglia neurons are guided to spinal targets by molecules differentially expressed along the dorso-ventral axis of the neural tube. NT-3-responsive muscle afferents project ventrally, cease extending, and branch upon contact with motoneurons (MNs), their synaptic partners. We have identified WNT-3 as a candidate molecule that regulates this process. Wnt-3 is expressed by MNs of the lateral motor column at the time when MNs form synapses with sensory neurons. WNT-3 increases branching and growth cone size while inhibiting axonal extension in NT-3- but not NGF-responsive axons. Ventral spinal cord secretes factors with axonal remodeling activity for NT-3-responsive neurons. This activity is present at limb levels and is blocked by a WNT antagonist. We propose that WNT-3, expressed by MNs, acts as a retrograde signal that controls terminal arborization of muscle afferents.

Afferent Pathways↗

PubMed2002

The LIM-only protein DRAL/FHL2 interacts with and is a corepressor for the promyelocytic leukemia zinc finger protein.

Members of the four-and-a-half-LIM domain (FHL) protein family, which are expressed in a tissue- and stage-specific manner, have been reported previously to function as transcriptional coactivators. One of these is the p53-inducible protein DRAL/FHL2 (where DRAL is down-regulated in rhabdomyosarcoma LIM domain protein). In this work, we identified potential binding partners for DRAL/FHL2 using an inducible yeast two-hybrid system. We present evidence of a functional interaction between the promyelocytic leukemia zinc finger protein (PLZF) and DRAL/FHL2. PLZF is a sequence-specific transcriptional repressor whose function relies on recruitment of corepressors that form part of the histone deacetylase complex involved in chromatin remodeling. DRAL/FHL2 interacts specifically with PLZF in vitro and in vivo and augments transcriptional repression mediated by PLZF. This is the first reported incidence of a bona fide FHL protein-mediated corepression and supports the notion of these proteins having a role as coregulators of tissue-specific gene expression.

Antigens, Polyomavirus Transforming↗

PubMed2002

Neutral endopeptidase is activated in cardiomyocytes in human aortic valve stenosis and heart failure.

BACKGROUND: The regulation of the cardiac neutral endopeptidase (EC 24.10, NEP) that degrades bradykinin and natriuretic peptides has been investigated in human cardiac hypertrophy and heart failure. Methods and Results- NEP mRNA was quantitated by real-time polymerase chain reaction (PCR) in left ventricular biopsies from patients with aortic valve stenosis (AS, n=19) and heart failure due to dilated cardiomyopathy (DCM, n=14), and control subjects with normal systolic function (CON, n=14). Left ventricular NEP mRNA content was increased 3-fold in AS (P<0.005) and 4.1-fold in DCM (P<0.002). The increase in NEP mRNA was related to the increase in end diastolic pressure in AS and DCM. In a second series, myocardial NEP enzymatic activity was determined. It increased 3.6-fold in AS (P<0.02) and 4-fold in DCM (P<0.002). NEP was localized in the myocardium by immunofluorescence microscopy and in situ PCR to myocytes and nonmyocyte areas and cells. CONCLUSIONS: Elevated cardiac NEP activity in pressure loaded and failing human hearts may increase the local degradation of bradykinin and natriuretic peptides.

Aortic Valve Stenosis↗

PubMed2002

Left but not right cardiac hypertrophy in atrial natriuretic peptide receptor-deficient mice is prevented by angiotensin type 1 receptor antagonist losartan.

Mice with a genetic deletion of the atrial natriuretic peptide (ANP) receptor, guanylyl cyclase A (GC-A -/-), have chronic arterial hypertension and cardiac hypertrophy from the first day of life. To characterize the role of the angiotensin II and endothelin systems in the development of this cardiovascular phenotype, the effects of chronic treatment with either the angiotensin type I (AT1) receptor antagonist losartan or the endothelin A receptor antagonist BSF208075 were tested. Losartan almost completely reversed systemic arterial hypertension and left ventricular hypertrophy of GC-A -/- mice. This was accompanied by a marked regression of the left ventricular mRNA expression of cardiac hypertrophy markers such as ANP and brain natriuretic peptide and a significant reduction of left ventricular and pulmonary interstitial collagen accumulation. BSF208075 had no effect on any of these cardiovascular parameters. Intriguingly, GC-A -/- mice also showed a very marked right ventricular hypertrophy, which was not reversed by losartan or BSF208075 treatment. Analyses of components of the renin-angiotensin system (RAS) revealed an inhibition of renal and systemic RAS contrasting with increased local left ventricular angiotensin II levels in GC-A -/- mice. Collectively, the results suggest that RAS plays a more important role than the endothelin system in the pathogenesis of arterial hypertension as well as left ventricular hypertrophy and fibrosis in GC-A gene-disrupted mice.

Angiotensin Receptor Antagonists↗

PubMed2002

Measles virus spreads in rat hippocampal neurons by cell-to-cell contact and in a polarized fashion.

Measles virus (MV) can infect the central nervous system and, in rare cases, causes subacute sclerosing panencephalitis, characterized by a progressive degeneration of neurons. The route of MV transmission in neurons was investigated in cultured rat hippocampal slices by using MV expressing green fluorescent protein. MV infected hippocampal neurons and spread unidirectionally, in a retrograde manner, from CA1 to CA3 pyramidal cells and from there to the dentate gyrus. Spreading of infection depended on cell-to-cell contact and occurred without any detectable release of infectious particles. The role of the viral proteins in the retrograde MV transmission was determined by investigating their sorting in infected pyramidal cells. MV glycoproteins, the fusion protein (F) and hemagglutinin (H), the matrix protein (M), and the phosphoprotein (P), which is part of the viral ribonucleoprotein complex, were all sorted to the dendrites. While M, P, and H proteins remained more intracellular, the F protein localized to prominent, spine-type domains at the surface of infected cells. The detected localization of MV proteins suggests that local microfusion events may be mediated by the F protein at sites of synaptic contacts and is consistent with a mechanism of retrograde transmission of MV infection.

Animals↗

PubMedDate not supplied

Design of artificial myocardial microtissues.

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

Animals↗