Connexin43 regulates joint location in zebrafish fins

AbstractJoints are essential for skeletal form and function, yet their development remains poorly understood. In zebrafish fins, joints form between the bony fin ray segments providing essentially unlimited opportunities to evaluate joint morphogenesis. Mutations in cx43 cause the short segment phenotype of short fin (sofb123) mutants, suggesting that direct cell–cell communication may regulate joint location. Interestingly, increased cx43 expression in the another long fin (alfdty86) mutant appears to cause joint failure typical of that mutant. Indeed, knockdown of cx43 in alfdty86 mutant fins rescues joint formation. Together, these data reveal a correlation between the level of Cx43 expression in the fin ray mesenchyme and the location of joints. Cx43 was also observed laterally in cells associated with developing joints. Confocal microscopy revealed that the Cx43 protein initially surrounds the membranes of ZNS5-positive joint cells, but at later stages becomes polarized toward the underlying Cx43-positive mesenchymal cells. One possibility is that communication between the Cx43-positive mesenchyme and the overlying ZNS5-positive cells regulates joint location, and upregulation of Cx43 in joint-forming cells contributes to joint morphogenesis

Etiology of pure tricuspid regurgitation based on anular circumference and leaflet area: Analysis of 45 necropsy patients with clinical and morphologic evidence of pure tricuspid regurgitation

Despite recent renewed interest in the detection of tricuspid valve regurgitation by echocardiographic and Doppler techniques, little morphologic information is available on dysfunctioning tricuspid valves. This report describes 45 necropsy patients with clinical and morphologic evidence of pure(no element of stenosis) tricuspid regurgitation and provides morphometric observations (anular circumference, leaflet area) of the tricuspid valve useful in determining the etiology of pure tricuspid regurgitation. Of 45 patients, 24 (53%) had pure tricuspid regurgitation resulting from an anatomically abnormal valve (prolap9e in 7, papillary muscle dysfunction in 6, rheumatic disease in 5, Ebstein's anomaly in 3, infective endocarditis in 2, carcinoid tumor in 1), and 21(47%) had an anatomically normal valve with systolic pulmonary artery hypertension (cor pulmonary in 12, mitral stenosis in 9). Anular circumference was dilated (> 12 cm) in patients with various causes of pulmonary hypertension, floppy valve and Ebstein's tricuspid anomaly. Leaflet area was increased in floppy valve and Ebstein's anomaly.Of the 45 patients, 24 had pulmonary systolic artery pressure measurements available for correlation with tricuspid valve morphology. Pulmonary artery pressures accurately predicted morphologically normal from abnormal valves in 16 patients (89 %). Morphologic overlap occurred in six patients with pulmonary pressures of 41 to 54 mm Hg. Of these six, the additional knowledge of normal or dilated anular circumference correctly separated valves with normal and abnormal leaflets

Activation of focal adhesion kinase by protein kinase Cϵ in neonatal rat ventricular myocytes

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2–30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 μmol/l; 1 h pretreatment). Adv-caPKCϵ, but not Adv-caPKCδ, activated FAK compared with a control Adv encoding β-galactosidase. Conversely, Adv-dnPKCϵ inhibited ET-induced FAK activation. Y-27632 (10 μmol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKCϵ-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 μmol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKCϵ-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 μmol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKCϵ, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src

The Cx43-like Connexin Protein Cx40.8 Is Differentially Localized during Fin Ontogeny and Fin Regeneration

Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration

Role of protein kinase C-ε in hypertrophy of cultured neonatal rat ventricular myocytes

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-ε, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-ε did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-ε also increased atrial natriuretic factor (ANF) and β-myosin heavy chain (MHC) mRNA levels 2.5 ± 0.3- and 2.1 ± 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector ( P < 0.05 for both). Conversely, Adv-dnPKC-ε did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of β-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-ε overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-ε activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin

Voltage dependent conductances are similar between Cx43 and Cx40.8-cd in transiently transfected N2a cells.

<p>(A) Transjunctional current family from a Cx43 expressing cell pair. V_j was varied ±180 mV in 30 mV steps. (B) Currents from a Cx40.8 expressing pair. (C) Normalized steady state G_j measures as a function of V_j show similar inactivation properties between the two constructs. Error bars are SEM.</p

Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.

<p>High resolution fluorescence microscopy was used to provide evidence for co-association of Cx43-mApple and Cx40.8-EGFP in common gap junction channels. Constructs that were co-transfected in HeLa cells are indicated to the left of the panels (A, B) <i>Homo sapiens</i> (Hs) Cx43-mApple + Hs Cx43-EGFP show uniformly yellow plaques, suggesting co-asociation. (C, D) Hs Cx43-mApple + Hs Cx26-EGFP show discrete green and red domains, revealing a lack of co-association. Arrows indicate green Hs Cx26-EGFP localization and arrowheads indicate red Hs Cx43-mApple localization. (E, F) <i>Danio rerio</i> (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.</p

Cartoon illustrating different domains of Cx43 and Cx40.8 used to generate protein chimeras.

<p>(A) Full length sequences of Cx43 and Cx40.8. The amino terminus through the end of the fourth transmembrane-spanning domain was not modified. Swaps of the entire carboxy termini were generated, as well as swaps between domains B/b, C/c, and D/d. Uppercase refers to the Cx43 domains; lowercase refers to the Cx40.8 domains. (B) Amino acid sequence of Cx43-B vs. Cx40.8-b domains.</p