Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

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Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1754-1763.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1754-1763

Author(s):

D S Crow ◽

E C Beyer ◽

D L Paul ◽

S S Kobe ◽

A F Lau

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Rous Sarcoma Virus ◽

Gap Junctional Communication ◽

Rous Sarcoma ◽

Junctional Communication ◽

Junction Protein ◽

Sarcoma Virus ◽

Gap Junction Protein ◽

Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

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Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment

AJP Cell Physiology ◽

10.1152/ajpcell.00345.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. C795-C804 ◽

Cited By ~ 41

Author(s):

Lucia Formigli ◽

Fabio Francini ◽

Alessia Tani ◽

Roberta Squecco ◽

Daniele Nosi ◽

...

Keyword(s):

Gap Junctions ◽

Lucifer Yellow ◽

Myoblast Differentiation ◽

Cell Contact ◽

Cx43 Expression ◽

Gap Junctional Communication ◽

Skeletal Myoblasts ◽

Junctional Communication ◽

Direct Cell ◽

Gap Junctional

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

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Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

The Journal of Cell Biology ◽

10.1083/jcb.111.5.2077 ◽

1990 ◽

Vol 111 (5) ◽

pp. 2077-2088 ◽

Cited By ~ 477

Author(s):

L S Musil ◽

B A Cunningham ◽

G M Edelman ◽

D A Goodenough

Keyword(s):

Cell Adhesion ◽

Gap Junctions ◽

Gap Junction ◽

Cell Lines ◽

Gap Junctional Communication ◽

Competent Cells ◽

Deficient Cell ◽

Junctional Communication ◽

Gap Junctional ◽

Cell Cell

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

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Myocardial gap junctions: targets for novel approaches in the prevention of life-threatening cardiac arrhythmias

Physiological Research ◽

10.33549/physiolres.931546 ◽

2008 ◽

pp. S1-S13

Author(s):

N Tribulová ◽

V Knezl ◽

Ľ Okruhlicová ◽

J Slezák

Keyword(s):

Gap Junctions ◽

Heart Function ◽

Cell Communication ◽

Persistent Atrial Fibrillation ◽

Gap Junctional Communication ◽

Novel Treatments ◽

Junctional Communication ◽

Life Threatening ◽

Direct Cell ◽

Gap Junctional

Direct cell-to-cell communication in the heart is maintained via gap junction channels composed of proteins termed connexins. Connexin channels ensure molecular and electrical signals propagation and hence are crucial in myocardial synchronization and heart function. Disease-induced gap junctions remodeling and/or an impairment or even block of intercellular communication due to acute pathological conditions results in derangements of myocardial conduction and synchronization. This is critical in the development of both ventricular fibrillation, which is a major cause of sudden cardiac death and persistent atrial fibrillation, most common arrhythmia in clinical practice often resulting in stroke. Many studies suggest that alterations in topology (remodeling), expression, phosphorylation and particularly function of connexin channels due to age or disease are implicated in the development of these life-threatening arrhythmias. It seems therefore challenging to examine whether compounds that could prevent or attenuate gap junctions remodeling and connexin channels dysfunction can protect the heart against arrhythmias that cause sudden death in humans. This assumption is supported by very recent findings showing that an increase of gap junctional conductance by specific peptides can prevents atrial conduction slowing or re-entrant ventricular tachycardia in ischemic heart. Suppression of ischemia-induced dephosphorylation of connexin seems to be one of the mechanisms involved. Another approach for identifying novel treatments is based on the hypothesis that even non-antiarrhythmic drugs with antiarrhythmic ability can modulate gap junctional communication and hence attenuate arrhythmogenic substrates.

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The role of gap junctions in amphibian development

Development ◽

10.1242/dev.89.supplement.365 ◽

1985 ◽

Vol 89 (Supplement) ◽

pp. 365-380

Author(s):

Anne E. Warner

Keyword(s):

Gap Junctions ◽

Protein Pattern ◽

Cell Communication ◽

Embryonic Cells ◽

Amphibian Embryo ◽

Junctional Communication ◽

Direct Cell ◽

Insect Epidermis ◽

Gap Junctional ◽

Communication Pathway

The possibility that communication through gap junctions may be important during embryonic development has often been raised since gap junctions were first described between early embryonic cells. It is now known that this direct cell-to-cell communication pathway disappears between groups of embryonic cells with different developmental fates as the embryo progresses through development, suggesting that transfer through the gap junctional pathway may play some part in controlling events during development. Supportive evidence for a role for gap junctions comes from experiments demonstrating that the properties of gap junctions differ at the border separating each segment in insect epidermis. Recently it has been shown that the ability to exchange small dyes between cells in the amphibian embryo depends on the position of each cell with respect to the grey crescent. When communication through gap junctions is prevented, by injecting antibodies to gap junctions protein, pattern formation is severely disturbed in the non-communicating region. The paper describes experiments on the pattern of junctional communication at early stages of development of the amphibian embryo and illustrates how anti-gap junction antibodies are being used to determine when and where communication through gap junctions may play an important role during development.

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Connexin mimetic peptides: specific inhibitors of gap-junctional intercellular communication

Biochemical Society Transactions ◽

10.1042/bst0290606 ◽

2001 ◽

Vol 29 (4) ◽

pp. 606-612 ◽

Cited By ~ 143

Author(s):

W. H. Evans ◽

S. Boitano

Keyword(s):

Gap Junctions ◽

Intercellular Communication ◽

Gap Junctional Intercellular Communication ◽

Reversible Inhibitors ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Wide Range ◽

Mimetic Peptides ◽

Gap Junctional ◽

Specific Sequences

Intercellular co-operation is a fundamental and widespread feature in tissues and organs. An important mechanism ensuring multicellular homoeostasis involves signalling between cells via gap junctions that directly connect the cytosolic contents of adjacent cells. Cell proliferation and intercellular communication across gap junctions are closely linked, and a number of pathologies in which communication is disrupted are known where connexins, the gap-junctional proteins, are modified. The proteins of gap junctions thus emerge as therapeutic targets inviting the development and exploitation of chemical tools and drugs that specifically influence intercellular communication. Connexin mimetic peptides that correspond to short specific sequences in the two extracellular loops of connexins are a class of benign, specific and reversible inhibitors of gap-junctional communication that have been studied recently in a broad range of cells, tissues and organs. This review summarizes the properties and uses of these short synthetic peptides, and compares their probable mechanism of action with those of a wide range of other less specific traditional gap-junction inhibitors.

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Gap junctions and fluid flow response in MC3T3-E1 cells

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1917 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1917-C1925 ◽

Cited By ~ 84

Author(s):

M. M. Saunders ◽

J. You ◽

J. E. Trosko ◽

H. Yamasaki ◽

Z. Li ◽

...

Keyword(s):

Fluid Flow ◽

Gap Junctions ◽

Bone Cell ◽

Dominant Negative ◽

Prostaglandin E ◽

Gap Junctional Intercellular Communication ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Text Response ◽

Gap Junctional

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca2+concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E2 (PGE2) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca2+ (Ca[Formula: see text]) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE2 but not to the Ca[Formula: see text] response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

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Involvement of connexin 43 phosphorylation and gap junctional communication between smooth muscle cells in vasopressin-induced ROCK-dependent vasoconstriction after hemorrhagic shock

AJP Cell Physiology ◽

10.1152/ajpcell.00258.2016 ◽

2017 ◽

Vol 313 (4) ◽

pp. C362-C370 ◽

Cited By ~ 9

Author(s):

Guangming Yang ◽

Xiaoyong Peng ◽

Yue Wu ◽

Tao Li ◽

Liangming Liu

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Gap Junctions ◽

Hemorrhagic Shock ◽

Connexin 43 ◽

Muscle Cells ◽

Gap Junctional Communication ◽

Contractile Effect ◽

Junctional Communication ◽

Gap Junctional

We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin 43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed that AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser262 and gap junctional communication in VSMCs. Activation of RhoA with U-46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junction independent. Gap junctions between VSMCs, gap junctional communication, and Cx43 phosphorylation at Ser262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process.

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Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.1.c147 ◽

2000 ◽

Vol 279 (1) ◽

pp. C147-C157 ◽

Cited By ~ 62

Author(s):

Kevin Ko ◽

Pamela Arora ◽

Wilson Lee ◽

Christopher McCulloch

Keyword(s):

Cell Adhesion ◽

Gap Junctions ◽

Adherens Junctions ◽

Functional Characterization ◽

Cell Monolayer ◽

Junctional Communication ◽

Donor Cells ◽

Gap Junctional ◽

Cell To Cell Adhesion ◽

Texas Red

Despite their significance in wound healing, little is known about the molecular determinants of cell-to-cell adhesion and gap junctional communication in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required Ca2+ and was >30-fold stronger than cell-to-fibronectin adhesion at 15 min. Electron micrographs showed rapid formation of adherens junction-like structures at ∼15 min that matured by ∼2–3 h; distinct gap junctional complexes were evident by ∼3 h. Immunoblotting showed that HGF expressed β-catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye transfer in a cohort of Texas red dextran-labeled cells, but this was almost completely abolished by the gap junction inhibitor β-glycyrrhetinic acid and the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model allows large numbers (>105) of cells to form synchronous cell-to-cell contacts, thereby enabling the simultaneous functional and molecular studies of adherens junctions and gap junctions.

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GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00635.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. H1952-H1964 ◽

Cited By ~ 12

Author(s):

Mathieu C. Rémond ◽

Grazia Iaffaldano ◽

Michael P. O'Quinn ◽

Nadejda V. Mezentseva ◽

Victor Garcia ◽

...

Keyword(s):

Reporter Gene ◽

Connexin 43 ◽

Ventricular Myocardium ◽

Phenotypic Heterogeneity ◽

Lacz Reporter ◽

Lacz Expression ◽

Heart Tube ◽

Junctional Communication ◽

Gap Junctional

This study examined transgenic mice whose expression of a β-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial β-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states.

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