scholarly journals Impaired Cx43 gap junction endocytosis causes cardiovascular defects in zebrafish

2021 ◽  
Author(s):  
Caitlin Hyland ◽  
Michael Mfarej ◽  
Giorgos Hiotis ◽  
Sabrina Lancaster ◽  
Noelle Novak ◽  
...  
Keyword(s):  
Gap Junction ◽  
Cardiac Tissue ◽  
Cultured Cells ◽  
Cell Communication ◽  
Cardiovascular Development ◽  
Zebrafish Model ◽  
Cx43 Protein ◽  
Junction Protein ◽  
Cardiovascular Defects ◽  
Electrical Impulses

AbstractGap junction proteins, termed connexins (Cx), mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting exchange of molecules, ions, and electrical impulses. The most ubiquitously expressed gap junction protein, connexin43 (Cx43) has been implicated in cardiovascular diseases including arrhythmias, cardiomyopathies, hypertension and diabetes. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43Δ256-289) and zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired cardiovascular development. These findings suggest that normal, unimpaired Cx43 gap junction endocytosis and turnover is an essential aspect of gap junction function as demonstrated here for cardiovascular development that when impaired can give rise to arrhythmias, heart malformations and aberrant vasculature structure and function.

scholarly journals Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

2021 ◽  
pp. mbc.E20-12-0797
Author(s):  
Caitlin Hyland ◽  
Michael Mfarej ◽  
Giorgos Hiotis ◽  
Sabrina Lancaster ◽  
Noelle Novak ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cardiac Tissue ◽  
Cultured Cells ◽  
Cell Communication ◽  
Cardiovascular Development ◽  
Zebrafish Model ◽  
Cx43 Protein ◽  
Molecular Events

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of connexins of only 1-5 hours, resulting in constant endocytosis and biosynthetic replacement of gap junction channels has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43∆256-289) and a zebrafish model ( cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx 43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that Cx43 gap junction endocytosis is an essential aspect of gap junction function and when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals AMP-Activated Protein Kinase α1 Regulates Cardiac Gap Junction Protein Connexin 43 and Electrical Remodeling Following Pressure Overload

2015 ◽  
Vol 35 (1) ◽  
pp. 406-418 ◽  
Author(s):  
Ioana Alesutan ◽  
Jakob Voelkl ◽  
Florian Stöckigt ◽  
Sobuj Mia ◽  
Martina Feger ◽  
...  
Keyword(s):  
Gap Junction ◽  
Connexin 43 ◽  
Cardiac Tissue ◽  
Pressure Overload ◽  
Protein Abundance ◽  
Wild Type ◽  
Cx43 Protein ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Amp Activated Protein Kinase

Background/Aims: Adenosine 5'-monophosphate (AMP)-activated protein kinase (Ampk) modulates a wide array of cellular functions and regulates various ion channels and transporters. In failing human hearts an increased Ampkα1 activity was observed. The present study aimed to uncover the impact of Ampkα1 on cardiac electrical remodeling. Methods: Gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and corresponding wild-type mice were exposed to pressure overload by “transverse aortic constriction” (TAC). In vivo electrophysiology was performed with a single catheter technique, myocardial conduction velocities and conduction characteristics investigated in isolated hearts, transcript levels quantified by RT-PCR and protein abundance determined by Western blotting. Moreover, connexin 43 (Cx43) was expressed in Xenopus oocytes with or without coexpression of wild-type or mutant AMPK and Cx43 protein abundance quantified utilizing confocal microscopy. Results: TAC treatment increased Ampkα1 protein expression in cardiac tissue from wild-type mice. TAC further increased left ventricular conduction inhomogeneity and triggered conduction blocks, effects blunted in the Ampkα1-/- mice. TAC treatment decreased Cx43 protein abundance in cardiac tissue, an effect significantly blunted in the Ampkα1-/- mice. TAC treatment did not modify Cx43 mRNA levels but increased ubiquitination of Cx43 protein, an effect mitigated by Ampkα1 deficiency. As shown in Xenopus oocytes, Cx43 cell membrane protein abundance was significantly downregulated by wild-type AMPKWT and constitutively active AMPKγR70Q, but not by catalytically inactive AMPKαK45R. Conclusion: Ampkα1 stimulates ubiquitination of the gap junction protein Cx43, thereby contributing to gap junction remodeling following pressure overload.

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 509-522
Author(s):  
R. Minkoff ◽  
S.B. Parker ◽  
E.L. Hertzberg
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Uniform Distribution ◽  
Rat Liver ◽  
Connexin 43 ◽  
Cell Communication ◽  
Exterior Surface ◽  
Junction Protein ◽  
Primary Palate ◽  
Gap Junction Protein

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 × 10(3) Mr, connexin 32) and heart (43 × 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 × 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the ‘heart’ 43 × 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

scholarly journals ERK acts in parallel to PKCδ to mediate the connexin43-dependent potentiation of Runx2 activity by FGF2 in MC3T3 osteoblasts

2012 ◽  
Vol 302 (7) ◽  
pp. C1035-C1044 ◽  
Author(s):  
Corinne Niger ◽  
Atum M. Buo ◽  
Carla Hebert ◽  
Brian T. Duggan ◽  
Mark S. Williams ◽  
...  
Keyword(s):  
Gap Junction ◽  
Cell Communication ◽  
Transcriptional Response ◽  
Luciferase Reporter ◽  
Western Blots ◽  
Erk Activation ◽  
Junctional Communication ◽  
Junction Protein ◽  
Reporter Assays ◽  
Cell Cell

The gap junction protein, connexin43 (Cx43), plays an important role in skeletal biology. Previously, we have shown that Cx43 can enhance the signaling and transcriptional response to fibroblast growth factor 2 (FGF2) in osteoblasts by increasing protein kinase C-δ (PKCδ) activation to affect Runx2 activity. In the present study, we show by luciferase reporter assays that the ERK signaling cascade acts in parallel to PKCδ to modulate Runx2 activity downstream of the Cx43-dependent amplification of FGF2 signaling. The PKCδ-independent activation of ERK by FGF2 was confirmed by Western blotting, as was the Cx43-dependent enhancement of ERK activation. Consistent with our prior observations for PKCδ, flow cytometry analyses show that Cx43 overexpression enhances the percentage of phospho-ERK-positive cells in response to FGF2, supporting the notion that shared signals among gap junction-coupled cells result in the enhanced response to FGF2. Western blots and luciferase reporter assays performed on osteoblasts cultured under low-density and high-density conditions revealed that cell-cell contacts are required for Cx43 to amplify ERK activation and gene transcription. Similarly, inhibition of gap junctional communication with the channel blocker 18β-glycyrrhetinic acid attenuates the Cx43-dependent enhancement of Runx2-transcriptional activity. In total, these data underscore the importance of cell-cell communication and activation of the ERK and PKCδ pathways in the coordination of the osteoblast response to FGF2 among populations of osteoblasts.

scholarly journals Complementary expression and phosphorylation of Cx46 and Cx50 during development and following gene deletion in mouse and in normal and orchitic mink testes

2015 ◽  
Vol 309 (3) ◽  
pp. R255-R276 ◽  
Author(s):  
R.-Marc Pelletier ◽  
Casimir D. Akpovi ◽  
Li Chen ◽  
Nalin M. Kumar ◽  
María L. Vitale
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Gap Junction ◽  
Sertoli Cell ◽  
Cell Communication ◽  
Seminiferous Tubules ◽  
Junction Protein ◽  
Mrna And Protein Expression ◽  
Autoimmune Orchitis ◽  
The Impact

Gap junction-mediated communication helps synchronize interconnected Sertoli cell activities. Besides, coordination of germ cell and Sertoli cell activities depends on gap junction-mediated Sertoli cell–germ cell communication. This report assesses mechanisms underlying the regulation of connexin 46 (Cx46) and Cx50 in mouse testis and those accompanying a “natural” seasonal and a pathological arrest of spermatogenesis, resulting from autoimmune orchitis (AIO) in mink. Furthermore, the impact of deleting Cx46 or Cx50 on the expression, phosphorylation of junction proteins, and spermatogenesis is evaluated. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. During the mink active spermatogenic phase, Cx50 became phosphorylated and localized to the site of the blood-testis barrier. By contrast, Cx46 was dephosphorylated and associated with annular junctions, suggesting phosphorylation/dephosphorylation of Cx46 and Cx50 involvement in the barrier dynamics. Cx46-positive annular junctions in contact with lipid droplets were found. Cx46 and Cx50 expression and localization were altered in mink with AIO. The deletion of Cx46 or Cx50 impacted on other connexin expression and phosphorylation and differently affected tight and adhering junction protein expression. The level of apoptosis, determined by ELISA, and a number of Apostain-labeled spermatocytes and spermatids/tubules were higher in mice lacking Cx46 ( Cx46−/−) than wild-type and Cx50−/− mice, arguing for life-sustaining Cx46 gap junction-mediated exchanges in late-stage germ cells secluded from the blood by the barrier. The data show that expression and phosphorylation of Cx46 and Cx50 are complementary in seminiferous tubules.

scholarly journals Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC

2007 ◽  
Vol 179 (6) ◽  
pp. 1301-1309 ◽  
Author(s):  
Joell L. Solan ◽  
Lucrecia Marquez-Rosado ◽  
Paul L. Sorgen ◽  
Perry J. Thornton ◽  
Philip R. Gafken ◽  
...  
Keyword(s):  
Gap Junction ◽  
Cultured Cells ◽  
Heart Tissue ◽  
Down Regulation ◽  
Stable Conformation ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Pkc Activation ◽  
Intercalated Disk ◽  
Regulatory Event

Phosphorylation at unspecified sites is known to regulate the life cycle (assembly, gating, and turnover) of the gap junction protein, Cx43. In this paper, we show that Cx43 is phosphorylated on S365 in cultured cells and heart tissue. Nuclear magnetic resonance structural studies of the C-terminal region of Cx43 with an S365D mutation indicate that it forms a different stable conformation than unphosphorylated wild-type Cx43. Immunolabeling with an antibody specific for Cx43 phosphorylated at S365 shows staining on gap junction structures in heart tissue that is lost upon hypoxia when Cx43 is no longer specifically localized to the intercalated disk. Efficient phosphorylation at S368, an important Cx43 channel regulatory event that increases during ischemia or PKC activation, depends on S365 being unphosphorylated. Thus, phosphorylation at S365 can serve a “gatekeeper” function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance.

scholarly journals Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src

1998 ◽  
Vol 140 (5) ◽  
pp. 1199-1209 ◽  
Author(s):  
Friso R. Postma ◽  
Trudi Hengeveld ◽  
Jacqueline Alblas ◽  
Ben N.G. Giepmans ◽  
Gerben C.M. Zondag ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
G Protein ◽  
Critical Role ◽  
Cell Communication ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled Receptor ◽  
Junction Protein ◽  
G Protein Coupled ◽  
Cell Cell

Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication.

scholarly journals Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes

1991 ◽  
Vol 273 (1) ◽  
pp. 67-72 ◽  
Author(s):  
D W Laird ◽  
K L Puranam ◽  
J P Revel
Keyword(s):  
Alkaline Phosphatase ◽  
Gap Junction ◽  
Cardiac Myocytes ◽  
Half Life ◽  
Cell Communication ◽  
Post Translational Modification ◽  
Phosphorylated Form ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Phosphate Groups

Cultured cardiomyocytes were used to study the turnover and post-translational modification of connexin43 (Cx43), a major gap junction protein in neonatal cardiac myocytes. Immunoprecipitation of [35S]Met-labelled lysates with anti-Cx43 antibodies followed by analysis using SDS/PAGE and fluorography revealed two bands, one at 40 kDa and the other at 42 kDa. Alkaline phosphatase treatment of [35S]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [32P]P1 incorporation into the 42 kDa band, but not into the band at 40 kDa. In addition, another alkaline phosphatase-sensitive phosphorylated form of Cx43 was identified at 44 kDa. In pulse-chase experiments, the half-life of Cx43 in cardiomyocytes was determined to be 1-2 h. Furthermore, the turnover rate of phosphate groups on Cx43 was found to be experimentally defined by the half-life of the protein. The observation that phosphate groups can remain with the protein throughout its life is consistent with the finding that in isolated adult rat heart gap junction plaques, Cx43 is primarily phosphorylated. We postulate that the rapid turnover of Cx43 and its multiple sites of phosphorylation play important roles in the regulation of cell-cell communication via gap junctions.

scholarly journals Increased expression of estrogen receptor beta in human uterine smooth muscle at term

2000 ◽  
pp. 92-99 ◽  
Author(s):  
JJ Wu ◽  
E Geimonen ◽  
J Andersen
Keyword(s):  
Estrogen Receptor ◽  
Cultured Cells ◽  
Estrogen Receptor Beta ◽  
Tissue Samples ◽  
Cx43 Protein ◽  
Protein Levels ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Low Levels ◽  
Receptor Beta

Expression of the cx43 gene for the gap junction protein, connexin43 (Cx43), through activator protein (AP)-1 activity has been shown to be inhibited in human primary myometrial cultures pretreated with estrogen. In the present study, the primary myometrial cultures were shown to express predominantly ERbeta, a subtype of estrogen receptor that inhibits AP-1 activity when bound to agonists. ERbeta levels were decreased in the primary myometrial cultures after treatment with the phorbol ester, 12-O-tetradecanolyl-13 acetate, to stimulate AP-1 activity, and this effect is inhibited if cells were pretreated with estrogen. Two isoforms of ERbeta were found in primary myometrial and leiomyoma cultured cells. Immunoblot and RT-PCR analyses indicated that ERbeta expression was increased in human term myometrial tissue compared with non-pregnancy tissue. Immunohistochemistry localized ERbeta to the nucleus in cells of term myometrial tissue samples that had high ERbeta expression. ERbeta was increased in term tissue in which Cx43 protein levels were low. In myometrial tissue in which Cx43 protein levels are greatest (e.g. during active labor), ERbeta was barely detectable. Only low levels of ERbeta were detected in non-pregnancy myometrial and leiomyoma tissues, and the lowest levels were found in tissues from mid cycle. In contrast, ERalpha was highly detectable in the non-pregnancy myometrial and leiomyoma tissues, but not in term myometrial tissue samples. This work indicates there is a dramatic switch from ERalpha to ERbeta expression in the myometrium during pregnancy. The results suggest that, during gestation, myometrial ERbeta may inhibit AP-1 activity and thus block induction of the cx43 gene and other labor-associated genes. Labor may ensue after a loss of myometrial ERbeta expression.

Protection afforded by ischemic preconditioning is not mediated by effects on cell-to-cell electrical coupling during myocardial ischemia-reperfusion

2003 ◽  
Vol 285 (5) ◽  
pp. H1909-H1916 ◽  
Author(s):  
Ferran Padilla ◽  
David Garcia-Dorado ◽  
Antonio Rodríguez-Sinovas ◽  
Marisol Ruiz-Meana ◽  
Javier Inserte ◽  
...  
Keyword(s):  
Gap Junction ◽  
Ischemic Preconditioning ◽  
Electrical Impedance ◽  
Ischemia Reperfusion ◽  
Electrical Coupling ◽  
Cell Communication ◽  
Tissue Impedance ◽  
Impulse Propagation ◽  
Junction Protein

The end-effectors of ischemic preconditioning (IPC) are not well known. It has been recently shown that transgenic mice underexpressing the gap junction protein connexin43 (Cx43) cannot be preconditioned. Because gap junctions allow spreading of cell death during ischemia-reperfusion in different tissues, including myocardium, we hypothesized that the protection afforded by IPC is mediated by effects on gap junction-mediated intercellular communication. To test this hypothesis, we analyzed the effect of IPC (5 min ischemia-5 min reperfusion × 2) on the changes in electrical impedance (four electrode probe) and impulse propagation velocity (transmembrane action potential) induced by ischemia (60 min) and reperfusion (60 min) in isolated rat hearts. IPC ( n = 8) reduced reperfusion-induced lactate dehydrogenase release by 65.8% with respect to control hearts ( n = 9) ( P = 0.04) but had no effect on the time of onset of rigor contracture (increase in diastolic tension), electrical uncoupling (sharp changes in tissue resistivity and phase angle in impedance recordings), or block of impulse propagation during ischemia. Normalization of electrical impedance during reperfusion was also unaffected by IPC. The lack of effect of IPC on ischemic rigor contracture and on changes in tissue impedance during ischemia-reperfusion were validated under in vivo conditions in pigs submitted to 48 min of coronary occlusion and 120 min of reperfusion. IPC ( n = 12) reduced infarct size (triphenyltetrazolium) by 64.9% ( P = 0.01) with respect to controls ( n = 17). We conclude that the protection afforded by IPC is not mediated by effects on electrical coupling. This result is consistent with recent findings suggesting that Cx43 could have effects on cell survival independent on changes in cell-to-cell communication.

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