Biomedical subjects

Sophie Clément

Publications and source records attributed to Sophie Clément.

9 recordsLinked to original sources

PubMed2006

Expression and function of alpha-smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation.

Three alpha-muscle actin isoforms are sequentially expressed during in vivo cardiac development. alpha-Smooth muscle actin is first and transiently expressed, followed by alpha-skeletal and finally alpha-cardiac actin. The significance of these transitions in actin gene expression during myogenesis remains to be determined. To understand whether actin isoforms have specific functions during cardiac development and cardiomyocyte contractility, we have hampered alpha-smooth muscle and alpha-skeletal actin expression and organization during embryonic stem cell differentiation towards cardiomyocyte. We show that the sequence of actin isoform expression displays similar pattern in the in vitro model and in mouse heart embryogenesis. Treatment with an interfering fusion peptide containing the N-terminal sequence of alpha-smooth muscle actin during a time window preceding spontaneous beating, prevents proper cardiac sarcomyogenesis, whereas alpha-skeletal actin-fusion peptide has no effect. Knockdown of alpha-smooth muscle actin in embryonic stem cells using RNA interference also affects cardiac differentiation. The application of both fusion peptides on beating embryoid bodies impairs frequency. These results suggest specific functional activities for actin isoforms in cardiogenesis and cardiomyocyte contractility.

Actins↗

PubMed2006

Correlation of alpha-skeletal actin expression, ventricular fibrosis and heart function with the degree of pressure overload cardiac hypertrophy in rats.

We have analysed alterations of alpha-skeletal actin expression and volume fraction of fibrosis in the ventricular myocardium and their functional counterpart in terms of arrhythmogenesis and haemodynamic variables, in rats with different degrees of compensated cardiac hypertrophy induced by infra-renal abdominal aortic coarctation. The following coarctation calibres were used: 1.3 (AC1.3 group), 0.7 (AC0.7) and 0.4 mm (AC0.4); age-matched rats were used as controls (C group). One month after surgery, spontaneous and sympathetic-induced ventricular arrhythmias were telemetrically recorded from conscious freely moving animals, and invasive haemodynamic measurements were performed in anaesthetized animals. After killing, subgroups of AC and C rats were used to evaluate in the left ventricle the expression and spatial distribution of alpha-skeletal actin and the amount of perivascular and interstitial fibrosis. As compared with C, all AC groups exhibited higher values of systolic pressure, ventricular weight and ventricular wall thickness. AC0.7 and AC0.4 rats also showed a larger amount of fibrosis and upregulation of alpha-skeletal actin expression associated with a higher vulnerability to ventricular arrhythmias (AC0.7 and AC0.4) and enhanced myocardial contractility (AC0.4). Our results illustrate the progressive changes in the extracellular matrix features accompanying early ventricular remodelling in response to different degrees of pressure overload that may be involved in the development of cardiac electrical instability. We also demonstrate for the first time a linear correlation between an increase in alpha-skeletal actin expression and the degree of compensated cardiac hypertrophy, possibly acting as an early compensatory mechanism to maintain normal mechanical performance.

Actins↗

PubMed2006

Development and evaluation of a modified colorimetric solid-phase microassay for measuring the activity of cellular and plasma (Factor XIII) transglutaminases.

Plasma TG (transglutaminase) [FXIII (Factor XIII)] stabilizes fibrin and plays an essential role in haemostasis. In the present paper, we report a simple colorimetric assay for measuring FXIII activity. The advantage of this approach, compared with all the other solid-phase assays described so far for measuring TG activity, is that the first substrate, namely the synthetic dipeptide, N-benzyloxycarbonyl-L-Gln-L-Gly, is coupled covalently by its C-terminus with amine-substituted polystyrene plates. Covalent coupling eliminates the problem of desorption of proteins such as casein and N,N'-dimethylcasein (first substrates) when 'blocking' buffers containing proteins, e.g. BSA, are employed, or when samples of plasma or cell homogenates are used. The principle of the assay itself is based on the incorporation of the well-known second substrate, 5-(biotinamido)pentylamine, into the gamma-carboxamide of glutamine in the immobilized dipeptide. The amount of biotinylated amine bound to the plate, as measured by the phosphatase activity of Extravidin phosphatase attached to the biotin moiety, is directly proportional to the TG activity. The method shows strong correlation (r = 0.96) with the radiometric assay for FXIII activity. For plasma samples, a linear response was obtained in the range 0-1.33 i.u. (international unit)/ml, versus the Behring standard. In a preliminary clinical investigation, the method was applied to normal and pathological plasma samples from patients with liver failure and disseminated intravascular coagulation. In the isolation of TG from guinea-pig liver, it was used to measure enzyme activity after each purification step. This method is rapid, sensitive and can easily be applied to routine clinical analyses and to specific research problems.

Animals↗

PubMed2005

The N-terminal Ac-EEED sequence plays a role in alpha-smooth-muscle actin incorporation into stress fibers.

We have previously shown that the N-terminal sequence AcEEED of alpha-smooth-muscle actin causes the loss of alpha-smooth-muscle actin from stress fibers and a decrease in cell contractility when introduced in myofibroblasts as a cell-penetrating fusion peptide. Here, we have investigated the function of this sequence on stress fiber organization in living cells, using enhanced green fluorescent protein (EGFP)-tagged alpha-smooth-muscle actin. The fusion peptide provokes the gradual disappearance of EGFP fluorescence of alpha-smooth-muscle actin from stress fibers and the formation of hitherto unknown rod-like structures. In addition to alpha-smooth-muscle actin, these structures contain cytoplasmic actins, gelsolin and cofilin but not other major actin-binding proteins. These rod-like structures are also visible in wild-type fibroblasts during normal cell spreading, suggesting that they represent a physiological step in the organization of alpha-smooth-muscle actin in stress fibers. Fluorescence-recovery-after-photobleaching experiments suggest that the fusion peptide reduces the dynamics of alpha-smooth-muscle actin and its incorporation in stress fibers. Here, we propose a new mechanism of how alpha-smooth-muscle actin is incorporated in stress fibers involving the sequence Ac-EEED.

Actins↗

PubMed2004

Biochemical evidence for interaction between smoothelin and filamentous actin.

The two major isoforms of smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled smoothelin, showed binding of smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.

Actins↗

PubMed2003

Alpha-actin isoform distribution in normal and failing human heart: a morphological, morphometric, and biochemical study.

We investigated the distribution of alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin isoforms in human heart during development, hypertrophy, and failure. At 20 weeks of fetal life, alpha-skeletal actin was localized in a small proportion of subendocardial and papillary muscle cardiomyocytes. At this gestation time, diffuse alpha-cardiac actin staining was observed, associated with focal expression of alpha-smooth muscle actin. In normal adult subjects, alpha-skeletal actin positive cardiomyocytes were distributed in a transmural gradient with the highest proportion located subendocardially. In myocardial hypertrophy and cardiomyopathies, the amount of alpha-skeletal actin was increased and diffuse staining was seen in all layers of ventricular myocardium, with the exception of idiopathic dilated cardiomyopathies. Cardiomyocytes were negative for alpha-smooth muscle actin in all pathological situations studied. As expected, fibroblasts in post-infarct scars expressed alpha-smooth muscle actin and transforming growth factor-beta1 but, surprisingly, were negative for these proteins in interstitial fibrosis. Our results demonstrate that increased expression of alpha-skeletal actin in the diseased human heart is associated with increased myocyte stretch, increased wall stress, and pressure overload, but not with idiopathic dilated cardiomyopathies. They also suggest that fibrotic changes develop with different mechanisms in scars versus interstitial fibrosis.

Actins↗

PubMed2002

Actin isoform pattern expression: a tool for the diagnosis and biological characterization of human rhabdomyosarcoma.

The diagnosis and characterization of rhabdomyosarcoma requires the use of combined histological and immunohistochemical criteria due to the variety of its histological patterns. The identification of actin isoform expression is accepted as a useful adjunct to the diagnosis and classification of soft tissue tumors. Using a new antibody specific for alpha-cardiac actin, obtained according to a recently described strategy for the production of polyclonal antibodies against actin isoforms [9], we have analyzed a series of 17 rhabdomyosarcomas, including all histological subtypes. In addition, we have evaluated the presence in these tumors of alpha-skeletal and alpha-smooth muscle actins. All specimens examined revealed a positive immunostaining for alpha-cardiac actin. Tumoral cells of eight cases also expressed alpha-smooth muscle actin and only three cases (all embryonal subtypes) were positive for alpha-skeletal actin. Our results indicate that immunohistochemical screening for alpha-cardiac actin expression is a useful tool for the diagnosis of rhabdomyosarcoma. They also suggest that the expression of alpha-skeletal actin is valuable in determining the subtype and possibly the state of differentiation of these tumors.

Actins↗

PubMed2002

High levels of cellular retinol binding protein-1 expression in leiomyosarcoma: possible implications for diagnostic evaluation.

Retinoid bioavailability is regulated by the activity of specific cytoplasmic receptors. High levels of cellular retinol binding protein-1 (CRBP-1) have been documented during experimental arterial and wound-healing processes, but data concerning neoplastic smooth muscle tissues are scarce and/or controversial. This study reports that the expression of CRBP-1 is markedly higher in uterine and gastrointestinal leiomyosarcomas than in leiomyomas and normal myometrium; CRBP-1 was practically absent in normal gastrointestinal smooth muscle tissue. CRBP-1 positivity was particularly elevated in the epithelioid variant of leiomyosarcoma; it was associated with increased proliferative and apoptotic rates and inversely related to smooth muscle differentiation evaluated by alpha- and gamma-smooth muscle actin and desmin expression. Western blotting and reverse-transcription polymerase chain reaction confirmed the observations concerning CRBP-1 and actin isoform expression and revealed higher NF-kappa-Bp65 and RAR alpha and lower Bax protein levels in leiomyosarcoma than in the other conditions. These findings document that a high CRBP-1 expression is associated with smooth muscle malignancy and suggest that CRBP-1 expression represents a new useful marker for the classification of unusual variants of smooth muscle tumors.

Adult↗

PubMed2002

Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of Renin genes.

Several studies have demonstrated that mice are polymorphic for the number of renin genes, with some inbred strains harboring one gene (Ren-1(c)) and other strains containing two genes (Ren-1(d) and Ren-2). In this study, the effects of 1% salt and deoxycorticosterone acetate (DOCA)/salt were investigated in one- and two-renin gene mice, for elucidation of the role of renin in the modulation of BP, cardiac, and renal responses to salt and DOCA. The results demonstrated that, under baseline conditions, mice with two renin genes exhibited 10-fold higher plasma renin activity, 100-fold higher plasma renin concentrations, elevated BP (which was angiotensin II-dependent), and an increased cardiac weight index, compared with one-renin gene mice (all P < 0.01). The presence of two renin genes markedly increased the BP, cardiac, and renal responses to salt. The number of renin genes also modulated the responses to DOCA/salt. In one-renin gene mice, DOCA/salt induced significant renal and cardiac hypertrophy (P < 0.01) even in the absence of any increase in BP. Treatment with losartan, an angiotensin II AT(1) receptor antagonist, decreased BP in two-renin gene mice but not in one-renin gene mice. However, losartan prevented the development of cardiac hypertrophy in both groups of mice. In conclusion, these data demonstrate that renin genes are important determinants of BP and of the responses to salt and DOCA in mice. The results confirm that the Ren-2 gene, which controls renin production mainly in the submaxillary gland, is physiologically active in mice and is not subject to the usual negative feedback control. Finally, these data provide further evidence that mineralocorticoids promote cardiac hypertrophy even in the absence of BP changes. This hypertrophic process is mediated in part by the activation of angiotensin II AT(1) receptors.

Animals↗