Biomedical subjects

Denise Paulin

Publications and source records attributed to Denise Paulin.

18 recordsLinked to original sources

PubMed2006

New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies.

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Animals↗

PubMed2005

Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart.

BACKGROUND: Serum response factor (SRF) is a cardiac transcription factor involved in cell growth and differentiation. We have shown, using the Cre/loxP system, that cardiac-specific disruption of SRF gene in the embryonic heart results in lethal cardiac defects. The role of SRF in adult heart is unknown. METHODS AND RESULTS: We disrupted SRF in the adult heart using a heart-specific tamoxifen-inducible Cre recombinase. This disruption led to impaired left ventricular function with reduced contractility, subsequently progressing to dilated cardiomyopathy, as demonstrated by serial echocardiography, including tissue Doppler imaging. The cytoarchitecture of cardiomyocytes was altered in the intercalated disks. All mutant mice died from heart failure 10 weeks after treatment. These functional and structural defects were preceded by early alterations in the cardiac gene expression program: major decreases in mRNA levels for cardiac alpha-actin, muscle creatine kinase, and calcium-handling genes. CONCLUSIONS: SRF is crucial for adult cardiac function and integrity. We suggest that the rapid progression to heart failure in SRF mutant mice results primarily from decreased expression of proteins involved in force generation and transmission, low levels of polymerized actin, and changes in cytoarchitecture, without hypertrophic compensation. These cardiac-specific SRF-deficient mice have the morphological and clinical features of acquired dilated cardiomyopathy in humans and may therefore be used as an inducible model of this disorder.

Animals↗

PubMed2005

Mechanism of binding of serum response factor to serum response element.

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124-245 fragment of serum response factor) with specific oligonucleotides from c-fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core-SRF labeled with bis-ANS showed that the protein is monomeric at low concentration (10(-7) m). The titration curves for the fluorescence anisotropy of fluorescein-labeled oligonucleotide revealed that under equilibrium conditions, the core-SRF monomers were bound sequentially to SRE at very low concentration (10(-9) m). Curve-fitting data showed also major differences between the wild-type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core-SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core-SRF was bound as a dimer to the wild-type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far-UV CD spectra indicated that the flexibility of core-SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein-labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA-protein interaction.

Amino Acid Sequence↗

PubMed2004

Desmin: a major intermediate filament protein essential for the structural integrity and function of muscle.

Desmin is a muscle-specific protein and a key subunit of the intermediate filament in cardiac, skeletal and smooth muscles. Desmin filaments are mainly located at the periphery of Z-disk of striated muscles and at the dense bodies of smooth muscle cells, and they have been postulated to play a critical role in the maintenance of structural and mechanical integrity of the contractile apparatus in muscle tissues. This review summarizes the findings in the regulation of the desmin gene and function of the desmin protein. The expression of desmin gene is regulated by a combination of different transcription control regions in muscle cells. The results from mice deficient in desmin reveal the fundamental role of desmin filaments in cell architecture, force transmission and mitochondrial function. Mice lacking desmin postnatally develop a dilated cardiomyopathy, a skeletal myopathy and smooth muscle defects. Some of desmin-related myopathies are attributable to a missense mutations and deletions in the desmin gene. Other desmin-related myopathies, in which the desmin gene is not mutated, could be related to mutations of the genes encoding the proteins that interact with desmin.

Animals↗

PubMed2004

Absence of dystrophin in mice reduces NO-dependent vascular function and vascular density: total recovery after a treatment with the aminoglycoside gentamicin.

OBJECTIVE: Mutations in the dystrophin gene causing Duchenne's muscular dystrophy (DMD) lead to premature stop codons. In mice lacking dystrophin (mdx mice), a model for DMD, these mutations can be suppressed by aminoglycosides such as gentamicin. Dystrophin plays a role in flow (shear stress)-mediated endothelium-dependent dilation (FMD) in arteries. We investigated the effect of gentamicin on vascular contractile and dilatory functions, vascular structure, and density in mdx mice. METHODS AND RESULTS: Isolated mice carotid and mesenteric resistance arteries were mounted in arteriographs allowing continuous diameter measurements. Mdx mice showed lower nitric oxide (NO)-dependent FMD and endothelial NO synthase (eNOS) expression as well as decreased vascular density in gracilis and cardiac muscles compared with control mice. Treatment with gentamycin restored these parameters. In contrast, smooth muscle-dependent contractions as well as endothelium-dependent or -independent dilation were not affected by dystrophin deficiency or by gentamicin treatment. CONCLUSIONS: Dystrophin deficiency induces a selective defect in flow-dependent mechanotransduction, thus attenuating FMD and eNOS expression, and may contribute to low arteriolar density. These findings open important perspectives regarding the mechanism involved in the pathophysiology of genetic diseases related to premature stop codons such as DMD.

Animals↗

PubMed2004

Desminopathies in muscle disease.

A recently identified class of myopathies is produced by abnormal desmin, and is characterized by a disorganization of the desmin filament network, the accumulation of insoluble desmin-containing aggregates, and destructive changes in the sarcomeric organization of striated muscles. The desmin filaments interact with various other cytoskeletal proteins. The distinct clinical phenotypes are heterogeneous, with progressive skeletal myopathy, cardiomyopathy, and respiratory insufficiency as the most prominent features. Most of the desmin mutations are autosomal dominant. Identification of the causal genetic mutations shows that the desmin gene is not the only gene implicated in desminopathies; other genes encoding desmin-associated proteins, such as alpha-B-crystallin, and synemin may also be involved. Patients with mutations in their alpha-B-crystallin gene, which produce similar skeletal and cardiac myopathies, also have opaque lenses. Knockout mice have helped to reveal the fundamental role of desmin filaments in cell architecture, sarcomere alignment, myofibril organization, and the distribution of mitochondria. Transgenic mice, which accumulate aggregates of desmin and associated proteins in their muscles, show that the loss of desmin intermediate function as a result of mutations in desmin itself, or in the desmin-associated constituents, is important for disease progression.

Animals↗

PubMed2004

Overcoming bacterial DNA contamination in real-time PCR and RT-PCR reactions for LacZ detection in cell therapy monitoring.

Since the introduction of the polymerase chain reaction the presence of contaminating bacterial DNA in the Taq polymerase preparations has hampered the use of this technique in microbiology. Lately, this inconvenience has equally impeded gene quantification in the field of cell or gene therapy, where bacterial genes such as LacZ are often used as tags to detect vectors or cells after their injection in the recipient organism. Several means to overcome the DNA contamination of Taq Polymerase have been reported with variable degrees of decontamination efficiency and alteration of the PCR reaction. Here we propose two protocols to efficiently quantify DNA or RNA from the LacZ gene by real-time PCR using either decontamination by low concentrations of DNAse I prior to PCR amplification or a highly purified Taq Polymerase which is devoid of detectable contamination.

Animals↗

PubMed2004

Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.

Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes.

Animals↗

PubMed2003

Temporal patterns of bone marrow cell differentiation following transplantation in doxorubicin-induced cardiomyopathy.

OBJECTIVE: Recent studies have suggested benefits of bone marrow cell transplantation for the regeneration of ischemic cardiac tissue. To extend the potential of cell transplantation, we assessed this treatment in a mouse model of acute nonischemic doxorubicin-induced cardiomyopathy. METHODS: To allow detection of engrafted cells, we used transgenic mice expressing the nuclear-located LacZ under the control of either desmin or vimentin promoters, which identify muscle lineage and mesenchymal cells, respectively. All transplanted cells were also labeled with the fluorescent dye DIL. One week after the administration of doxorubicin (15 mg/kg), mice were intramyocardially injected with either allogeneic unpurified bone marrow cells (6 x 10(6) in 30 microl, n=59) or purified sca-1(pos) cells (4 x 10(5) in 30 microl, n=22). In parallel, control normal mice received only unpurified bone marrow cells (n=28). Hearts were harvested at serial intervals until 2 weeks after transplantation and analyzed by immunohistochemistry to assess the degree of engraftment and transplanted cell differentiation. RESULTS: In control mice, no differentiation of bone marrow cells was detected. In contrast, unpurified bone marrow cells grafted into diseased myocardium featured two successive phases of cell differentiation. The first yielded cells with a mesenchymal phenotype (44.1+/-10.1 cells/3 x 10(-2) mm(3) at 2 days), was transient and lasted 1 week. The second phase was characterized by cells with a muscular phenotype detected in a small number of cells (5.6+/-2.3 cells/3 x 10(-2) mm(3) at 7 days). Two weeks after transplantation, some of these cells appeared phenotypically close to cardiomyocytes, as evidenced by morphology and positive staining for myosin binding protein C, vinculin and myosin heavy chain. In sca-1(pos) hematopoietic progenitor grafted mice hearts, no transdifferentiation into cardiac cells was detected at any time point. CONCLUSION: These data support the hypothesis of the potential for a myogenic differentiation of bone marrow cells following engraftment in a nonischemic model of global cardiomyopathy. Bone marrow-derived cells amenable to cardiac differentiation are present in total unpurified bone marrow but not in the sca-1(pos) hematopoietic progenitor cell population. However, the very small number of transdifferentiated cells raises concerns over their functional efficacy.

Animals↗

PubMed2003

Expression of the intermediate filament protein synemin in myofibrillar myopathies and other muscle diseases.

Synemin is a member of the intermediate protein superfamily. Previous studies in avian and rodent skeletal and cardiac muscles have demonstrated that synemin localises at the Z-band, where it associates with desmin and alpha-actinin. In the present study, the distribution of synemin was examined using immunohistochemistry in muscle biopsy specimens from patients suffering from myofibrillar myopathy (MM, n=6), dermatomyositis (DM, n=3), inclusion body myositis (IBM, n=5), oculopharyngeal muscular dystrophy (OPD, n=3) and denervation atrophy (DA, n=3), to investigate the possible participation of this protein in the pathogenesis of various muscular diseases. Of patients affected by MM, two showed the presence of mutations in the desmin gene; none had mutations in the alphaB-crystallin gene; and no mutations were identified in synemin or syncoilin genes of three patients. Synemin immunohistochemistry disclosed a faint staining corresponding to the Z-bands in the cytoplasm of control muscle fibres; in contrast, focal aggregates of synemin were seen in patients with MM. Increased synemin immunoreactivity was identified diffusely or in the subsarcolemmal space of scattered fibres in patients with DM, and in vacuolated fibres of patients with IBM and OPD. Strong synemin immunoreactivity was observed in target formations and atrophic fibres of patients with denervating disorders, as well as in atrophic fibres, regardless of their origin, in all patients studied. Synemin co-localised with desmin, as seen on consecutive serial sections immunostained with anti-synemin or anti-desmin antibodies. These observations demonstrate abnormal accumulations containing both synemin and desmin in muscle fibres in patients with MM, IBM, DM, OPD and DA. Considering the important role of synemin as one of intermediate filaments of skeletal and cardiac muscle, its destruction and accumulation in the intracellular debris suggest that synemin may participate in the pathogenesis of these disorders.

Adult↗

PubMed2003

Synemin expression in developing normal and pathological human retina and lens.

Synemin (Syn) is an intermediate filament (IF) protein. To gain insight into a morphogenetic role of Syn, we have studied its expression patterns in the developing human retina and lens and compared it with those of other IF proteins. In addition, we have tested Syn expression in fetuses (23 and 28 weeks) affected by Walker-Warburg syndrome (WWS), Meckel syndrome, and trisomy 13. In the retina, Syn expression starts in the nerve fiber and ganglion cell layers (NFL and GCL) at 15 weeks, remains there in up to 20 weeks, and spreads to other layers and may be colocalized with vimentin, GFA, or neurofilaments in the subsequent 16 weeks. This expansion of Synemin expression from 20 to 28 weeks is not observed in WWS in which Syn immunoreactivity in NFL is reduced and Vim expression is increased. Changes are seen in Syn or vimentin expressions in the retinae of 23-week-old Meckel syndrome or 28-week-old trisomy 13 fetuses. Syn expression in the lens is, at first (16 weeks), uniformly distributed, becoming stronger in the epithelium of the anterior part at 25 weeks and later. As in the retina, Syn expression in lens is also selectively affected in WWS. The colocalization of Synemin with vimentin, GFA, or NF supports the idea that Syn is a key cross-linking protein that connects different cytoskeletal structures. Moreover, stagnant Syn expression in WWS retina and lens reinforces the notion of a significant role of this protein in morphogenesis.

Fetal Diseases↗

PubMed2002

Differences in the activation of the GFAP gene promoter by prion and viral infections.

The expression of glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. After surgical injury or viral infections, an increase in this protein reflects reactive gliosis in the brain. We analyzed the activation of the GFAP gene in transgenic mice using a prion and two different viruses (rabies and Theiler viruses). Inoculation of the transgenic mice with the C506M3 mouse prion strain resulted in activation of the GFAP-lacZ transgene. Expression of the GFAP transgene increased concomitantly with the expression of GFAP in astrocytes from the infected mice. In contrast, infection with rabies or Theiler's virus had no effect on the expression of the GFAP transgene, showing that the glial reactions to these infectious agents involved different mechanisms. These findings indicate that the activation of the endogenous GFAP gene as a consequence of viral infection could involve different regulatory pathways than activation as a result of prion infection. The first 2 kb upstream from the start codon of the GFAP gene seems to provide enough activation domains to produce efficient activation of the reporter gene in prion-infected mice.

Animals↗

PubMed2002

Excessive microvascular adaptation to changes in blood flow in mice lacking gene encoding for desmin.

OBJECTIVE: Desmin, an intermediate filament, has a key role in the integrity of myocytes, and its absence induces cardiomyopathies. Mice lacking desmin (Des-/- group) exhibit microvascular dysfunction leading to smooth muscle hyporeactivity. We investigated the effect of the absence of desmin in mice (Des-/- mice versus Des+/+ mice) on the adaptation of mesenteric arteries to changes in blood flow. METHODS AND RESULTS: With the use of selective ligations of second-order mesenteric arteries, blood flow was either diminished (low flow [LF]) or elevated (high flow [HF]); respective LF to HF values were 136+/-18 to 206+/-29 microL/min for Des+/+ mice and 119+/-14 to 189+/-24 microL/min for Des-/-mice in daughter arteries. Two weeks after ligation, arteries were mounted in an arteriograph, allowing the measurement of diameter under controlled conditions of pressure and flow. In HF arteries, diameter changes in response to increases in pressure were higher in Des-/- mice than in Des+/+ mice. Conversely, in LF arteries, diameter was lower in Des-/- mice. Flow-dependent dilation was higher in HF arteries and lower in LF arteries than in control arteries. This adaptation was lower in Des-/- mice than in Des+/+ mice (11.6+/-3.1% versus 25.5+/-4.8% dilation, respectively). Endothelial NO synthase expression increased in HF arteries in both strains. CONCLUSIONS: These findings provide a demonstration of the role of the intermediate filament desmin in microvascular remodeling. This dysfunction might take place in desmin-related myopathies.

Adaptation, Physiological↗

PubMed2002

Comparative study of immune responses induced after immunization with plasmids encoding the HIV-1 Nef protein under the control of the CMV-IE or the muscle-specific desmin promoter.

The objective of this study was to compare immune responses induced against HIV-1 Nef after DNA immunization with Nef encoding plasmids under the control of an ubiquitous or a muscle-specific promoter. To this end, plasmids containing HIV-1 nef under the control of the Cytomegalovirus or the human desmin promoters, specifically expressed in muscle cells, were constructed. Different groups of BALB/c mice were immunized with 10 or 100 microg of both constructs, controls were the pcDNA3 vector or the Nef protein in Freund's adjuvant (Nef-CFA). Our data showed that both plasmids stimulated anti-Nef humoral responses in a dose-dependent manner. The anti-Nef antibody response, however, was earlier and higher with the CMV-IE promoter than with the desmin promoter. We also showed that Nef expressing plasmids induced high titers of anti-Nef antibodies (10(4)), comparable to those obtained in the Nef-CFA group (4x10(4)). Analysis of the specificity of anti-Nef antibodies revealed no influence of the promoter, and in contrast to Nef-CFA, plasmid immunization elicited anti-Nef antibodies directed principally against conformational-dependent epitopes. Data on the lymphoproliferative response showed that the specificity of expression, or the plasmid dose, did not affect the onset-time or the intensity of the response. The predominant IgG2a isotype of anti-Nef antibodies and the cytokine profile, mostly IL-2 and IFN-gamma, produced by Nef-stimulated spleen cells indicated a Th1 response in plasmid-immunized mice, in contrast to mice immunized with Nef-CFA, where a Th2 response was induced. In conclusion, these data indicate that antigen expression by muscle cells is sufficient to stimulate a Th1 immune response.

AIDS Vaccines↗

PubMed2002

Mechanical function of intermediate filaments in arteries of different size examined using desmin deficient mice.

Protein composition and mechanical function of intermediate filaments were examined in arteries of different sizes using desmin deficient mice (Des-/-) and their wild-type controls (Des+/+). Using SDS-PAGE gels and Western blots we found a gradient in desmin expression in the arterial tree; the desmin content increased from the elastic artery aorta, via the muscular mesenteric artery to the resistance-sized mesenteric microarteries approximately 150 microm in diameter in Des+/+ mice. Mechanical experiments were performed on the aorta, the mesenteric artery and resistance-sized arteries using wire myographs. For aorta and mesenteric artery, no differences in passive or active circumference- stress relations were found between Des-/- and Des+/+ mice. In microarteries, both passive and active stress were lower in the Des-/- group. In conclusion, large elastic and muscular arteries contain a relatively low amount of desmin, and the desmin intermediate filaments do not seem to play a major role in the mechanical properties of these larger arterial vessels. In the microarteries, where expression of desmin is high, desmin plays a role in supporting both passive and active tension.

Animals↗

PubMed2002

Analysis of mural cell recruitment to tumor vessels.

BACKGROUND: Tumor blood vessels are both structurally and functionally abnormal compared with normal vessels. A limited support of mural cells may contribute to these abnormalities. Here, we characterized mural cell recruitment in 2 mouse tumor models and addressed the question of why tumor vessels fail to recruit a proper coat of mural cells. METHODS AND RESULTS: We studied mural cell recruitment to the vasculature of 2 transplantable mouse tumor models, T241 fibrosarcoma and KRIB osteosarcoma. We found that both tumors formed a vessel network with heterogeneous and highly abnormal organization of mural cells. Transplantation of tumors to mice expressing lacZ in mural cells demonstrated that these cells were host-derived. Although tumor vessel endothelium expressed PDGF-B, an embryonic mitogen for mural cells, only very few PDGFRbeta-positive cells were found to be associated with the developing tumor vasculature, suggesting a limited pool of recruitable mural cells. We tested whether exogenous mural cells could be recruited to tumor vessels by injecting mixtures of T241 tumor cells and embryonic mesenchymal cells isolated from mice expressing lacZ in mural cells. In the tumors that arose, lacZ-positive cells were efficiently recruited to the tumor vessels. CONCLUSIONS: T241 and KRIB tumors show a similar highly abnormal organization of vessel-associated mural cells. T241 tumor vessels seem highly capable of recruiting exogenously added mural cells. The sparse mural cell coat of tumor vessels may result from a limited pool of mural cells available for recruitment.

Actins↗

PubMed2002

High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.

Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration.

Animals↗

PubMed2001

Selective microvascular dysfunction in mice lacking the gene encoding for desmin.

The intermediate filament desmin has a key role in the integrity and contractility of skeletal and cardiac myocytes. Its absence or aggregation leads to cardiomyopathies. In arteries desmin is distributed heterogeneously; vascular disorders might also occur in its absence. We studied endothelial and muscular functions in arteries from mice lacking desmin (des-/-), compared with control (des+/+). Carotid and mesenteric resistance arteries were mounted in vitro in arteriographs. Desmin was located exclusively in smooth muscle cells. In arteries from des-/- mice, pressure-induced (myogenic) tone was unchanged, but agonist-induced tone decreased in resistance arteries (no change in large arteries). Flow (shear stress)- and acetylcholine-induced, endothelium-dependent dilation, as well as endothelium-independent dilation, were also decreased in resistance arteries. To our knowledge, this is the first study of vascular contractile and dilatory functions in arteries lacking desmin. Although vascular reactivity was normal in large arteries, it decreased strongly in small resistance arteries. Thus, desmin is required in vascular smooth muscle cells and in resistance arteries, for efficient control of vascular tone and consequently for an optimal blood flow supply. This microvascular defect found in the absence of desmin might play a major role in myopathies seen in desmin-related diseases.

Animals↗