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Biomedical subjects

Y Capetanaki

Publications and source records attributed to Y Capetanaki.

At least 19 recordsLinked to original sources
PubMed2006

Blood vessels and desmin control the positioning of nuclei in skeletal muscle fibers.

Skeletal muscle fibers contain hundreds to thousands of nuclei which lie immediately under the plasmalemma and are spaced out along the fiber, except for a small cluster of specialized nuclei at the neuromuscular junction. How the nuclei attain their positions along the fiber is not understood. Here we show that the nuclei are preferentially localized near blood vessels (BV), particularly in slow-twitch, oxidative fibers. Thus, in rat soleus muscle fibers, 81% of the nuclei appear next to BV. Lack of desmin markedly perturbs the distribution of nuclei along the fibers but does not prevent their close association with BV. Consistent with a role for desmin in the spacing of nuclei, we show that denervation affects the organization of desmin filaments as well as the distribution of nuclei. During chronic stimulation of denervated muscles, new BV form, along which muscle nuclei align themselves. We conclude that the positioning of nuclei along muscle fibers is plastic and that BV and desmin intermediate filaments each play a distinct role in the control of this positioning.

Animals↗
PubMed2001

High-throughput site-directed mutagenesis in ES cells.

Introduction of nonselectable mutations into the genome of embryonic stem cells by homologous recombination allows to investigate the function of genes at the molecular level and has been achieved, however, at very low efficiencies by the Hit and Run, Tag and Exchange, and Double Replacement strategies. Comparing those strategies at a single locus with vectors derived from a single fragment of the desmin gene led to the improvement of two strategies by employing a new selection cassette and modified selection procedures. Modified strategies resulted in the introduction of nonselectable point-mutations in 53% of the Hit and Run derived embryonic stem cell clones and in 0.7% of the Tag and Exchange clones. Efficiency of intrachromosomal recombination at Hit alleles outscored replacement-type recombination at the tagged alleles making the modified Hit and Run strategy the method of choice for the efficient introduction of nonselectable point mutations into the genome of embryonic stem cells.

Alleles↗
PubMed2001

Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm.

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.

Cell Differentiation↗
PubMed2001

Desmin integrates the three-dimensional mechanical properties of muscles.

Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.

Animals↗
PubMed2001

Paranemin and the organization of desmin filament networks.

De novo expression of vimentin, GFAP or peripherin leads to the assembly of an extended intermediate filament network in intermediate filament-free SW13/cl.2 cells. Desmin, in contrast, does not form extended filament networks in either SW13/cl.2 or intermediate filament-free mouse fibroblasts. Rather, desmin formed short thickened filamentous structures and prominent spot-like cytoplasmic aggregates that were composed of densely packed 9-11 nm diameter filaments. Analysis of stably transfected cell lines indicates that the inability of desmin to form extended networks is not due to a difference in the level of transgene expression. Nestin, paranemin and synemin are large intermediate filament proteins that coassemble with desmin in muscle cells. Although each of these large intermediate filament proteins colocalized with desmin when coexpressed in SW-13 cells, expression of paranemin, but not synemin or nestin, led to the formation of an extended desmin network. A similar rescue of desmin network organization was observed when desmin was coexpressed with vimentin, which coassembles with desmin, or with keratins, which formed a distinct filament network. These studies demonstrate that desmin filaments differ in their organizational properties from the other vimentin-like intermediate filament proteins and appear to depend upon coassembly with paranemin, at least when they are expressed in non-muscle cells, in order to form an extended filament network.

Avian Proteins↗
PubMed2001

Sarcomere number regulation maintained after immobilization in desmin-null mouse skeletal muscle.

The serial sarcomere number of skeletal muscle changes in response to chronic length perturbation. The role of the intermediate filament desmin in regulating these changes was investigated by comparing the architectural adaptations of the tibialis anterior, extensor digitorum longus (EDL) and soleus from wild-type mice with those of homozygous desmin knockout mice after hindlimb immobilization. After 28 days, serial sarcomere number increased significantly in the lengthened wild-type tibialis anterior (by approximately 9%) and EDL (by approximately 17%). Surprisingly, muscles from desmin knockout mice also experienced significant serial remodeling, with the serial sarcomere number of the tibialis anterior increasing by approximately 10% and that of the EDL by approximately 27%. A consistent result was observed in the shortened soleus: a significant decrease in sarcomere number was observed in the muscles from both wild-type (approximately 26%) and knockout (approximately 12%) mice. Thus, although desmin is not essential for sarcomerogenesis or sarcomere subtraction in mouse hindlimb muscles, the results do suggest subtle differences in the nature of sarcomere number adaptation. We speculate that desmin may play a role in regulating the optimal arrangement of sarcomeres within the muscle or in sensing the magnitude of the immobilization effect itself.

Animals↗
PubMed2000

Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function.

Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADP-stimulated mitochondrial respiration in situ using saponin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADP-stimulated oxygen consumption and the dissociation constant (K(m)) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.

Adenosine Diphosphate↗
PubMed2000

Desmin knockout muscles generate lower stress and are less vulnerable to injury compared with wild-type muscles.

The functional role of the skeletal muscle intermediate filament system was investigated by measuring the magnitude of muscle force loss after cyclic eccentric contraction (EC) in normal and desmin null mouse extensor digitorum longus muscles. Isometric stress generated was significantly greater in wild-type (313 +/- 8 kPa) compared with knockout muscles (276 +/- 13 kPa) before EC (P < 0.05), but 1 h after 10 ECs, both muscle types generated identical levels of stress ( approximately 250 kPa), suggesting less injury to the knockout. Differences in injury susceptibility were not explained by the different absolute stress levels imposed on wild-type versus knockout muscles (determined by testing older muscles) or by differences in fiber length or mechanical energy absorbed. Morphometric analysis of longitudinal electron micrographs indicated that Z disks from knockout muscles were more staggered (0.36 +/- 0. 03 microm) compared with wild-type muscles (0.22 +/- 0.03 microm), which may indicate that the knockout cytoskeleton is more compliant. These data demonstrate that lack of the intermediate filament system decreases isometric stress production and that the desmin knockout muscle is less vulnerable to mechanical injury.

Age Factors↗
PubMed1999

The absence of desmin leads to cardiomyocyte hypertrophy and cardiac dilation with compromised systolic function.

Desmin is the muscle-specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. There have been increasing reports of human cardiomyopathies associated with abnormal accumulation and aggregation of desmin filaments. Recently identified desmin mutations in humans suffering from skeletal muscle myopathy and cardiomyopathy suggest that these diseases might arise as a consequence of impaired function of desmin filaments. Previous generation of desmin null mice in our laboratory demonstrated that the absence of desmin results in myocyte ultrastructural defects and myocyte cell death leading to fibrosis and calcification of the myocardium. However, the effects that these defects have on cardiac function were not addressed. To further our understanding of desmin function in vivo, and in order to address the direct involvement of desmin in cardiomyopathy, we investigated the effect of the absence of desmin on myocardial mass, myocyte size and shape, changes in gene expression and cardiac systolic and diastolic function in mice. Morphometric characterization of isolated cardiomyocytes demonstrated a 24% increase in cell volume in the desmin null mice, solely due to an increase in transverse section area, suggesting for the first time that mice lacking the intermediate filament protein desmin develop concentric cardiomyocyte hypertrophy. This type of hypertrophy was accompanied by induction of embryonic gene expression and later by ventricular dilatation, and compromised systolic function. These results demonstrate that desmin is essential for normal cardiac function, and they suggest that the absence of an intact desmin filament system, rather than accumulation of the protein, may be responsible for the pathology seen in some of the desmin associated cardiomyopathies.

Animals↗
PubMed1997

Desmin in muscle formation and maintenance: knockouts and consequences.

Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earliest known myogenic markers in both skeletal muscle and heart. Its expression precedes that of all known muscle proteins including the members of the MyoD family of myogenic helix-loop-helix (mHLH) regulators with the exception of myf5. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. In vitro studies using both antisense RNA and homologous recombination techniques in embryonic stem (ES) cells demonstrated that desmin plays a crucial role during myogenesis, as inhibition of desmin expression blocked myoblast fusion and myotube formation. Both in C2C12 cells and differentiating embryoid bodies, the absence of desmin interferes with the normal myogenic program, as manifested by the inhibition of the mHLH transcription regulators. To investigate the function of desmin in all muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, a considerable number of these mice are viable and fertile, potentially due to compensation by vimentin, nestin or synemin. However, desmin null mice demonstrate a multisystem disorder involving cardiac, skeletal and smooth muscle, beginning early in their postnatal life. Histological and electron microscopic analysis in both heart and skeletal muscle tissues reveals severe disruption of muscle architecture and degeneration. Structural abnormalities include loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondrial number and organization, and loss of nuclear shape and positioning. Loose cell adhesion and increased intercellular space are prominent defects. The consequences of these abnormalities are most severe in the heart, which exhibits progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. There is a direct correlation between severity of damage and muscle usage, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. In conclusion, the studies so far have demonstrated that though desmin is absolutely necessary for muscle differentiation in vitro, muscle development can take place in vivo in the absence of this intermediate filament protein. However, desmin seems to play an essential role in the maintenance of myofibril, myofiber and whole muscle tissue structural and functional integrity.

Animals↗
PubMed1996

A single MEF2 site governs desmin transcription in both heart and skeletal muscle during mouse embryogenesis.

Desmin, the muscle-specific intermediate filament protein is one of the earliest known myogenic makers both in heart and in somites. We have previously shown that high levels of desmin expression in the skeletal cell line C2C12 are due to a distal enhancer, which contains a muscle-specific factor-(2MEF2) binding site, adjacent to an E box, the binding site of the myogenic Helix-Loop-Helix (mHLH) regulators. We have further shown that MEF2C, a myocyte restricted member of the MEF2 family and all four mHLH factors can bind to their corresponding sites and through a cooperation with a second proximal E box can transactivate the desmin promoter. To study the significance of these regulatory elements in vivo, we have generated transgenic mice with desmin-lacZ reporters, intact or mutated at the MEF2 and E box of the enhancer. We show that the cis-acting DNA sequences within the 1-kb 5' flanking region of the mouse desmin gene are sufficient to direct appropriate temporal transcription both in heart and in skeletal muscle during mouse embryogenesis. Mutation at the MEF2 site completely suppressed transcription of the linked lacZ transgene in both developing heart and somites of the embryos. Mutation of the E box only suppressed activation in skeletal muscle precursors (somites and limb buds) but not in cardiac muscle. These data demonstrate that the MEF2 site is indispensable for the desmin enhancer function both in heart and in skeletal muscle. In addition, MEF2 cooperation with the mHLH regulators is absolutely necessary for proper transcriptional activity during embryonic skeletal muscle development.

Animals↗
PubMed1996

Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.

Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils and abnormal mitochondrial organization. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. The present data demonstrate the essential role of desmin in the maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity, and show that the absence of desmin leads to muscle degeneration.

Animals↗
PubMed1995

Cytoskeletal control of myogenesis: a desmin null mutation blocks the myogenic pathway during embryonic stem cell differentiation.

A differentiating system based on embryonic stem (ES) cell-derived embryoid bodies (EBs) which recapitulates the in vivo cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation.

Animals↗
PubMed1994

An E box in the desmin promoter cooperates with the E box and MEF-2 sites of a distal enhancer to direct muscle-specific transcription.

The first 85 nt upstream of the transcription initiation site of the mouse desmin gene, which contain an E box (E1), the binding site of the helix-loop-helix myogenic regulators, are sufficient to confer low level muscle-specific expression. High levels of desmin expression are due to an enhancer, located between nucleotides -798 and -976, which contains an additional E box (E2) and a muscle-specific enhancer factor-2 (MEF-2) binding site. We have previously shown that both myoD and myogenin can bind to the proximal (E1) and distal (E2) boxes. Here we demonstrate that MEF-2C, a myocyte-restricted member of the MEF-2 family, can bind to the desmin MEF-2 site. Functional units for the enhancer activity required intact E2 and MEF-2 elements. The desmin enhancer can function relatively well with either the E2 box or the MEF-2 site and only mutation of both eliminates transcriptional enhancement; the presence of both of these elements is required for maximum enhancer activity. On the other hand, mutagenesis of just the proximal E1 box showed that this element is essential for desmin gene expression. Double mutations of E1 with E2 or MEF-2 sites suggested that, to achieve high levels of desmin gene expression, E1 serves most possibly as an intermediary for either E2 or MEF-2 enhancer elements to function. The location of the E1 site relative to the TATA box is crucial. Its activity is DNA turn- and distance-dependent. Furthermore, this box seems to be the main element for desmin transactivation by myoD and myogenin in 10T1/2 cells. Its inactivation diminishes the transactivation by these factors; MRF4 and Myf5, however, can still partially function, possibly by using the distal E2 box.

Animals↗
PubMed1994

Inhibition of desmin expression blocks myoblast fusion and interferes with the myogenic regulators MyoD and myogenin.

The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.

Amino Acid Sequence↗
PubMed1994

Biosynthesis of glycosphingolipids is reduced in the absence of a vimentin intermediate filament network.

Our previous observations on the immunocytochemical colocalization of intermediate filaments and glycosphingolipids led us to analyze the role of filaments in the biosynthesis and intracellular transport of glycosphingolipids. Cells with (vim+) and without (vim-) vimentin intermediate filaments were cloned from the adrenal carcinoma cell line SW13. There was no difference between vim+ and vim- cells in the proportion of newly synthesized C6-NBD-glucosylceramide transported to the plasma membrane. The vim+ cells synthesized glycosphingolipids, especially lactosylceramide and globotriosylceramide, and to a lesser extent GM3 ganglioside, more rapidly than vim- cells. The altered rate of biosynthesis did not result from differences in the levels of the glycosyltransferases that synthesize those compounds. To determine whether the presence of a vimentin network was responsible for the differences in biosynthesis, mouse vimentin cDNA was transfected into vim- cells. Transfected cells that expressed a mouse vimentin network demonstrated a twofold or greater increase in the rate of biosynthesis of neutral glycosphingolipids and gangliosides. There was no difference between vim+ and vim- cells in the synthesis of ceramide or sphingomyelin, or in their content of phospholipids or cholesterol. The nature of the biochemical defect(s) underlying the diminished incorporation of radiolabeled sugars into glycosphingolipids is unclear. Possibilities include alterations in the ultrastructure of the Golgi and/or abnormalities in a portion of the endocytic pathway.

Adrenal Cortex Neoplasms↗