Biomedical subjects

D B Gros

Publications and source records attributed to D B Gros.

6 recordsLinked to original sources

PubMed2001

Inhibition of endothelial wound repair by dominant negative connexin inhibitors.

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-betaGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ~50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Animals↗

PubMed1999

Downregulation of connexin 45 gene products during mouse heart development.

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.

Amino Acid Sequence↗

PubMed1999

Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle.

OBJECTIVE: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.

Animals↗

PubMed1996

Connexins in mammalian heart function.

In heart, the propagation of electrical activity is mediated by intercellular channels, referred to as junctional channels, aggregated into gap junctions and localised between myocytes. These channels consist of structurally related transmembrane proteins, the connexins, three of which (CX43, CX40 and CX45) have been shown to be associated with the myocytes of mammalian heart; a fourth, CX37, was detected exclusively in endothelial cells. In this paper, we review the recent data dealing with the topographical heterogeneity of expression of these connexins in the different cardiac tissues and the unique conductance properties of the channels they form, and attempt to assess the role played by each connexin and the consequences of their multiplicity in the propagation of action potentials.

Action Potentials↗

PubMed1985

The Mr 28,000 gap junction proteins from rat heart and liver are different but related.

The sequence of the amino-terminal 32 residues of the rat heart Mr 28,000 gap junction protein presented here allows, for the first time, a sequence comparison of gap junctional proteins from different tissues (heart and liver). Comparison of the rat heart gap junction protein sequence and that available from rat liver reveals 43% sequence identity and conservative changes at an additional 25% of the positions. Both proteins exhibit a hydrophobic domain which could represent a transmembrane span of the junction. This result unequivocally demonstrates the existence of at least two forms of the gap junction protein. As yet, no homology is evident between the gap junctional proteins of either heart or liver and main intrinsic protein from rat eye lens.

Amino Acid Sequence↗

PubMed1983

Comparative analysis of the gap junction protein from rat heart and liver: is there a tissue specificity of gap junctions?

Gap junctions have been isolated from both rat heart and liver, tissues where junctions are typical in appearance and physiology. The purity of the fractions obtained was monitored by electron microscopy (thin-sectioning and negative staining) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The myocardial gap junctions are comprised of a single polypeptide of Mr 28,000, apparently derived from a protein of Mr 30,000. Hepatic gap junctions are also comprised of a single native protein of Mr 28,000 as previously reported. Exhaustive trypsin digestion of the isolated junctions cleaves both of these proteins similarly, while leaving their characteristic junctional lattice structures intact. However, comparison of heart and liver junctional proteins by two-dimensional peptide mapping of tryptic and alpha-chymotryptic fragments, followed by high pressure liquid chromatography, reveals no homology between these proteins.

Animals↗