Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative fluorescent confocal images of Cx40-YFP, V85I-YFP and L221I-YFP (top panels). The overlaid fluorescent images on top of phase contrast images are also shown (bottom panels). All three constructs were able to form gap junction plaque-like structures at the cell-cell junction. Scale bar = 10 µm. ( B ) Voltage steps of 20 mV were applied to one cell of a transfected N2A cell pair and the junctional current (I j ) was recorded in the second cell. There was no significant difference between the I j in cell pairs expressing Cx40-YFP, V85I-YFP or L221I-YFP. The junctional conductance (G j ) was calculated and there was no significant difference between the G j of cell pairs expressing Cx40-YFP, V85I-YFP or L221I-YFP.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Construct, Transfection, Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative images of propidium iodide (PI)-uptake under divalent cation-free (DCF) conditions for isolated, individual, transfected HeLa cells. Successful transfection can be identified by their tagged green/yellow fluorescent proteins (green colour in the first column images). PI-uptake (red colour in the second column images) can be seen in cells expressing Cx26-GFP, V85I-YFP and L221I-YFP, but no uptake was seen in cells expressing YFP alone or Cx40-YFP. Scale bar = 20 µm. ( B ) Quantification of PI-uptake under DCF conditions. V85I-YFP (67.6%, n = 10) and L221I-YFP (83.2%, n = 10) showed a significant increase in PI-uptake compared to Cx40-YFP (4.6%, n = 14, ***indicates P<0.001). Other AF-linked Cx40 mutants, Q49X, L229M and I75F, were also studied and did not show any PI-uptake.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Isolation, Transfection, Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Comparison of PI-uptake for divalent cation containing (DCC) and divalent cation-free (DCF) conditions. The PI-uptake for cells expressing Cx26-GFP, V85I-YFP and L221I-YFP was significantly increased under DCF conditions compared to DCC conditions. Also the addition of the hemichannel blocker carbenoxolone (CBX, 100 µM) under DCF conditions significantly decreased PI-uptake. ( B ) [Ca 2+ ] o dose-dependent PI-uptake. Cx26-GFP, V85I-YFP and L221I-YFP all showed [Ca 2+ ] o dependent PI-uptake. Cx40-YFP did not show PI-uptake for any concentrations tested.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative confocal images showing the anti-Cx40 antibody localizations of untagged Cx40, V85I and L221I expressed in HeLa (top panels) and N2A (bottom panels) cells. In both cell lines, Cx40, V85I and L221I were all able to traffic to the cell-cell interface and form gap junction plaque-like structures. Scale bar = 10 µm. ( B ) There was no significant difference between the G j of cell pairs expressing Cx40, V85I or L221I. ( C ) Co-expression of Cx40V85I-IRES-GFP or L221I-IRES-GFP with Cx43-mRFP (V85I+Cx43 or L221I+Cx43) in N2A cell pairs showed a similar G j with cell pairs expressing Cx43-mRFP. The number of cell pairs are indicated on the bars.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Untagged V85I and L221I showed a significant increase in PI-uptake compared to both wild-type Cx40 and Cx43 under the divalent cation-free (DCF) conditions. ( B ) The addition of divalent cations (DCC, open bars) or CBX (100 µM, gray bars) blocked the PI-uptake from cells expressing Cx43, V85I and L221I. However, the addition of the pannexin 1 channel blocker probenecid (200 µM, hatched bars) did not affect PI-uptake. ( C ) PI-uptake in cells expressing untagged V85I or L221I under DCF were significantly (P<0.001 in both cases) reduced by the addition of flufenamic acid (FFA, 50 µM) or mefloquine (MFQ, 25 µM). The total number of experiments are indicated on the bar.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative confocal images of PI-uptake for HeLa cells transfected with wild-type Cx40 or Cx40 mutants (V85I or L221I). Time points of 0, 4 and 20 minutes of incubation with PI are displayed. During 20 minutes of incubation with PI, only cells expressing the V85I and L221I mutants showed PI uptake. Scale bar = 50 µm. ( B ) Ratio of current PI fluorescence intensity over the initial baseline fluorescence over a 20 minute incubation. L221I showed the fastest rate of PI-uptake, with V85I having a slightly slower rate of uptake, however, both L221I and V85I had similar levels of PI-uptake near the end of 20 minute incubation. The addition of 100 µM CBX blocked PI-uptake. Cx40 expressing cells failed to show any PI-uptake.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Transfection, Incubation, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: Voltage clamp ramp protocol (−40 to 110 mV) was used to study currents under divalent cation containing saline (DCC, black traces) and the divalent-cation free saline (DCF, red traces). Putative hemichannel currents (the current amplitude differences between red and black traces) were observed in cells expressing AF-linked Cx40 mutants, V85I (A), L221I (B) and wild-type Cx43 (C, left panel). Most of Cx40-expressing cells failed to show the current (C, right panel). The DCF-dependent currents in the mutant-expressing cells were largely blocked by carbenoxolone (CBX, 100 µM, green traces). Bar graph summarized the percentages of cells displayed hemichannel current during the voltage ramp under DCF conditions (D). The connexin constructs expressed and the numbers of cells recorded are indicated. Note only 4/33 cells expressing Cx40 displayed putative hemichannel current (data not shown).

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing, Mutagenesis, Construct

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: AF-linked germline Cx40 mutants have been shown to impair gap junction function via impaired localization (Q49X) or channel function (I75F). Dominant negative on Cx40 (Q49X and I75F) and/or transdominant negative actions on Cx43 were also observed (Q49X, I75F and L229M) , . Present study showed that AF-linked Cx40 mutants, V85I and L221I, increased hemichannel function.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Dominant Negative Mutation

Journal: Frontiers in Physiology

Article Title: Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

doi: 10.3389/fphys.2018.01383

Figure Lengend Snippet: Atrial profibrotic and connexin remodeling. (A) Representative Western blot examples (top) and quantification of protein expression (bottom; mean ± SEM) of periostin, vimentin, α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP9), transforming growth factor-β1 (TGF-β1), fibronectin and collagen 1α (col1a) in right-atrial tissue homogenates from Ctl (white bars), HFrEF (blue bars) or HFrEF-cAF (red/blue-striped bars) patients. Vertical white lines separate non-adjacent lanes on the same blot. (B) Representative Western blot examples (top) and quantification of protein expression (bottom; mean ± SEM) of total connexin-40 (Cx40), total and Ser368-phosphorylated connexin-43 (Cx43). GAPDH was used as loading control and is shown for the samples used for Western blots of periostin, vimentin, α-SMA and MMP9, for TGF-β1, and for fibronectin and col1a. Numbers in bars indicate number of patients. ∗ indicates P < 0.05 vs. Ctl, # indicates P < 0.05 vs. HFrEF.

Article Snippet: Protein levels of α smooth-muscle actin (αSMA, 1:500, A5228, Sigma-Aldrich, St. Louis, MO, United States), calsequestrin (CSQ, 1:2,500, PA1-913, Thermo Fisher Scientific), collagen 1α (Col1a, 1:1,000, sc-293182, Santa Cruz Biotechnology, Santa Cruz, CA, United States), connexin-40 (Cx40, 1:1,000, ab1726, Merck Millipore, Burlington, MA, United States), total and Ser368-phosphorylated connexin-43 (Cx43, 1:1,000, 3511, Cell Signaling Technology, Danvers, MA, United States), fibronectin (1:1,000, sc-8422, Santa Cruz Biotechnology), GAPDH (1:20,000, 5G4 6C5, HyTest, Turku, Finland), junctin (1:2,000, LS-C196703, LifeSpan BioSciences, Seattle, WA, United States), junctophilin-2 (1:1,000, sc-134875, Santa Cruz), matrix metallopeptidase 9 (MMP9, 1:200, ab38898, Abcam, Cambridge, United Kingdom), Na + -Ca 2+ -exchanger type-1 (NCX1, 1:1,000, R3F1, Swant, Marly, Switzerland), periostin (1:1,000, sc-134875, Santa Cruz Biotechnology), total, Ser16- and Thr17-phosphorylated phospholamban (PLB, all 1:1,000, ab2865 and ab92697, Abcam, Cambridge, United Kingdom and A010-13, Badrilla Ltd., Leeds, United Kingdom), total ryanodine receptor type-2 channel (RyR2, 1:1,000, MA3-916, Thermo Fisher Scientific), sarcolipin (1:100, ABT13, Merck Millipore), sarcoplasmic reticulum (SR) Ca 2+ -ATPase type-2a (SERCA2a; 1:2,000, sc-8095, Santa Cruz Biotechnology), transforming growth factor β1 (TGF-β1, 1:1000, ab9758, Abcam), and vimentin (1:1,000, sc373717, Santa Cruz Biotechnology) were determined using appropriate primary antibodies.

Techniques: Western Blot, Expressing

Journal: The FASEB Journal

Article Title: Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure

doi: 10.1096/fj.201700699R

Figure Lengend Snippet: Cx43 protein is increased in CCM3KD brain endothelial cells. A) Control or CCM3KD (siRNA-mediated 85% CCM3 knockdown; Supplemental Fig. 1) whole-cell lysates were prepared and analyzed via Western blot. Cx43 protein, but not Cx37 or Cx40 protein, is significantly elevated relative to control (n = 3 independent experiments). *P < 0.05 compared to control. B) RNA extracted from control and CCM3KD cells and subjected to reverse-transcription quantitative PCR revealed no significant changes in Cx43 transcript. Data represent means ± sem fold-change normalized to β-actin (Cx43: n = 6, Cx40, Cx37: n = 3 independent experiments). **P < 0.01 compared with control. C) Whole-cell lysates were subjected to cell fractionation. Cx43 was only present in the insoluble fraction (caveolin-1) and not in the cytosol (Hsp70; representative Western blot, n = 3 independent experiments). D) FRAP analysis of control and CCM3KD cells expressing Cx43-AcGFP. FRAP was performed on membrane ROIs (n = 6 ROIs from 6 cells). E) Immunofluorescence staining of Cx43 (column 1), merged Cx43, and DAPI staining (column 2), and zoom images of Cx43 GJs along the cell membrane (column 3, dashed lines) in control and CCM3KD cells (GJ plaques are indicated with white arrows). Scale bar, 100 μm. F) Cx43 GJ plaque size was measured in ImageJ. For 3 image fields with ∼8 cells/field, the entire membrane region of every cell was selected to analyze particle (GJ plaque) size in Cx43-labeled samples. Average plaque size is reported as mean ± sem of all GJ plaques measured (∼50 plaques/field). This analysis revealed significantly larger GJ plaques in CCM3KD cells compared with controls. **P < 0.01.

Article Snippet: Primary Abs used were as follows: Cx37 (Abcam, Cambridge, MA, USA); Cx40 (GeneTex, Irvine, CA, USA); Cx43 and β-tubulin; CCM1 (Boster Bio, Pleasanton, CA, USA); CCM2 (Sigma-Aldrich); CCM3 (ProteinTech, Rosemont, IL, USA); and ZO-1 and Claudin-5 (Thermo Fisher Scientific).

Techniques: Control, Knockdown, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Fractionation, Expressing, Membrane, Immunofluorescence, Staining, Labeling