Abstract 13799: Electrical Pacing Inhibits Gap Junction-Mediated Cardiac Cell -Cell Communication by Promoting Cx43-Acetylation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Valerio Azzimato ◽  
Viviana Meraviglia ◽  
Claudia Colussi ◽  
Maria Cristina Florio ◽  
Alice Panariti ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Gap Junction ◽  
Cell Communication ◽  
Neonatal Rat ◽  
Cell Protein ◽  
Cardiac Cell ◽  
Cx43 Expression ◽  
Rat Cardiomyocytes ◽  
Electrical Pacing ◽  
Cell Cell

Background: Communication among cardiomyocytes depends upon Gap Junction (GJ) protein expression and conductance. Previous studies demonstrated that electrical stimulation can induce GJ remodeling and evidences from neurons also indicate that electrical pacing modifies Lysine acetylase (KAT) and deacetylases (KDAC) activities. Objectives: Aim of the present work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation dependent mechanisms. Methods and Results: Neonatal rat cardiomyocytes (NRCM; n=3) and in HL-1 atrial cells (n=20) were exposed to electrical field stimulation for 24 hours (Ionoptix C-Pace®; 0.5 Hz, 20 V, 0.5 msec pulses). Connexin 43 (Cx43) expression decreased almost 50% in NRCM and 40 % in HL-1; in contrast Cx40 and Cx45 expression was unchanged. Further, confocal microscopy revealed that electrical stimulation induced Cx43 accumulation in the cytoplasm of HL-1 cells. Electrical stimulation significatly down-regulated KDAC activity up to the 30% (n=3), whereas KAT activity was not modified; the net effect was a general increase of cell protein acetylation, confirmed by western blot analysis. Specifically, the pacing-dependent acetylation of Cx43 was proven by immunoprecipitation assay (n=5). Interestingly, our model mimicked the action of the KDAC pan-inhibitors TSA and SAHA on Cx43 expression and intracellular distribution, although we did not observe Cx43 mRNA significant reduction in electrically stimulated cells. In agreement, MG132 proteasome inhibitor (10 μM) restored Cx43 expression level. Finally, also the treatment of paced cells with the KAT inhibitor Anacardic Acid (0.5 μM) was able to rescue Cx43 level (n=4). Intriguingly, preliminary results also indicate lateralization and increased acetylation of Cx43 in the left ventricles of dogs with pacing-induced dilated cardiomyopathy (n=2). Conclusions: In vitro electrical stimulation of cardiac cells promotes Cx43 acetylation, which results in Cx43 down-modulation and intracellular relocalization. These findings suggest that electrical activity-dependent increase in acetylation may represent a novel mechanism for the regulation of cardiomyocyte communication.

Abstract 16737: Inhibition of Tgf-β1 Signaling Protect Against Cell-Cell Decoupling and Asynchronous Automaticity of Tbx18-ipms

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Jinqi Fan ◽  
Nam Kyun Kim ◽  
Natasha Fernandez ◽  
Minji Shin ◽  
Hee Cho
Keyword(s):  
Gap Junction ◽  
Protein Level ◽  
Electrical Coupling ◽  
Neonatal Rat ◽  
Electrical Remodeling ◽  
Tgfβ Signaling ◽  
Cx43 Expression ◽  
Intramyocardial Injection ◽  
Tgfβ Receptors ◽  
Cell Cell

Background: We have previously demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells (TBX18-iPMs). However, the nascent automaticity appeared to wane over time, characterized by loss of syncytial pacing but preservation of single cell automaticity. Hypothesis: Based on increase in Tgfβ ligands in TBX18-iPMs and the known role of Tgfβ signaling in electrical remodeling, we hypothesized that loss of syncytial automaticity is due to electrical decoupling of the iPMs mediated by Tgfβ signaling. Methods: Adenoviral vectors expressing either human TBX18 or GFP were used for gene transfer into neonatal rat ventricular myocytes (NRVMs) or adult rat ventricular myocardium in vivo. Results: NRVM monolayers transduced with GFP were mostly quiescent, interspersed by paroxysmal contractions. In contrast, TBX18 transduced NRVM monolayers showed spontaneous and rhythmic contractions, but the syncytial pacing wavered over the next 5-7 days although numerous iPMs continued to beat asynchronously. Treatment of TBX18-iPMs with an inhibitor of Tgfβ receptors, A83-01, preserved syncytial pacing, suggesting that electrical remodeling cues from Tgfβ signaling led to loss of iPM-iPM electrical coupling. We examined the molecules that are integral to the function of gap junction in the myocardium, namely Cx43, N-cadherin and β-catenin. Cx43 and N-cadherin were downregulated in TBX18-iPMs compared to control by 77±16% and 43±11% (p<0.01, n=6), respectively. Total β-catenin protein level was unaltered, but its distribution decreased at the sarcolemmal and increased in the nuclei of TBX18-iPMs compared to control. Inhibition of Tgfβ with A83-01 restored Cx43 protein level in TBX18-iPMs (2.2-folds higher) compared to untreated TBX18-iPMs. Similarly, adult rats that received intramyocardial injection of TBX18 showed increased Cx43 expression at the boundary between the iPMs and the host myocardium when the animals were treated with systemic delivery of A83-01 for one week compared to TBX18 animals treated with DMSO. Conclusions: TBX18 activates Tgfβ signaling which suppresses cell-cell electrical coupling. Inhibition of Tgfβ signaling in TBX18-iPMs preserves gap junction components and syncytial pacing.

scholarly journals Transforming growth factor-β1-induced N-cadherin drives cell–cell communication through connexin43 in osteoblast lineage

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yueyi Yang ◽  
Wenjing Liu ◽  
JieYa Wei ◽  
Yujia Cui ◽  
Demao Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Gap Junction ◽  
Cell Line ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Cx43 Expression ◽  
Mc3t3 Cell ◽  
Primary Osteoblasts ◽  
Tgf Β1 ◽  
Cell Cell

AbstractGap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-β1(TGF-β1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-β1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-β1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell–cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-β1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-β1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.

scholarly journals IL-6 loss causes ventricular dysfunction, fibrosis, reduced capillary density, and dramatically alters the cell populations of the developing and adult heart

2009 ◽  
Vol 296 (5) ◽  
pp. H1694-H1704 ◽  
Author(s):  
Indroneal Banerjee ◽  
John W. Fuseler ◽  
Arti R. Intwala ◽  
Troy A. Baudino
Keyword(s):  
Cardiac Function ◽  
Cardiac Fibroblasts ◽  
Cell Communication ◽  
Cell Types ◽  
Capillary Density ◽  
Cell Populations ◽  
Cardiac Cell ◽  
Altered Expression ◽  
Cell Cell

Interleukin-6 (IL-6) is a pleiotropic cytokine responsible for many different processes including the regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs, including the heart. Recent studies have indicated that IL-6 is a critical component in the cell-cell communication between myocytes and cardiac fibroblasts. In this study, we examined the effects of IL-6 deficiency on the cardiac cell populations, cardiac function, and interactions between the cells of the heart, specifically cardiac fibroblasts and myocytes. To examine the effects of IL-6 loss on cardiac function, we used the IL-6 −/− mouse. IL-6 deficiency caused severe cardiac dilatation, increased accumulation of interstitial collagen, and altered expression of the adhesion protein periostin. In addition, flow cytometric analyses demonstrated dramatic alterations in the cardiac cell populations of IL-6 −/− mice compared with wild-type littermates. We observed a marked increase in the cardiac fibroblast population in IL-6 −/− mice, whereas a concomitant decrease was observed in the other cardiac cell populations examined. Moreover, we observed increased cell proliferation and apoptosis in the developing IL-6 −/− heart. Additionally, we observed a significant decrease in the capillary density of IL-6 −/− hearts. To elucidate the role of IL-6 in the interactions between cardiac fibroblasts and myocytes, we performed in vitro studies and demonstrated that IL-6 deficiency attenuated the activation of the STAT3 pathway and VEGF production. Taken together, these data demonstrate that a loss of IL-6 causes cardiac dysfunction by shifting the cardiac cell populations, altering the extracellular matrix, and disrupting critical cell-cell interactions.

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.

Medium perfusion enables engineering of compact and contractile cardiac tissue

2004 ◽  
Vol 286 (2) ◽  
pp. H507-H516 ◽  
Author(s):  
Milica Radisic ◽  
Liming Yang ◽  
Jan Boublik ◽  
Richard J. Cohen ◽  
Robert Langer ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Troponin I ◽  
Capture Rate ◽  
Initial Density ◽  
Neonatal Rat ◽  
Excitation Threshold ◽  
Interstitial Flow ◽  
Rat Cardiomyocytes ◽  
Gap Junctional

We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 × 108 cells/cm3) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric α-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter × 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-μm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.

scholarly journals Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell–Cell Communication?

2021 ◽  
Vol 22 (17) ◽  
pp. 9169
Author(s):  
Camillo Peracchia
Keyword(s):  
Gap Junction ◽  
Genetic Diseases ◽  
Channel Gating ◽  
Cell Communication ◽  
Potential Effect ◽  
Gap Junction Channel ◽  
Direct Cell ◽  
Connexin Genes ◽  
Cell Cell

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

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 Flow cytometry analysis of gap junction-mediated cell–cell communication: Advantages and pitfalls

2006 ◽  
Vol 69A (6) ◽  
pp. 487-493 ◽  
Author(s):  
Paula Candida Fonseca ◽  
Oscar Kenji Nihei ◽  
Wilson Savino ◽  
David C. Spray ◽  
Luiz Anastacio Alves
Keyword(s):  
Flow Cytometry ◽  
Gap Junction ◽  
Cell Communication ◽  
Flow Cytometry Analysis ◽  
Cell Cell
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