Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of full length Cx43 and αCT1 peptide. B) αCT1 interaction with ZO-1 PDZ domains as indicated by EDC zero-length cross-linking to GST fusion PDZ1, PDZ2 and PDZ3 polypeptides and neutravidin labeling of biotin-tagged peptide at concentrations of 5, 25 and 50 μM. The deletion of the CT Isoleucine (I) in αCT1-I renders this peptide incompetent to interact with the ZO-1 PDZ2 domain. C) Coomassie blue gel of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT and PKC-ε, with (αCT1) and without (Vehicle) αCT1. The fainter band above GST-Cx43 bands (indicated by lines) in the αCT1 lanes were cut from gels and analyzed by Tandem Mass Spectrometry (MS/MS). The boxes to right of gel show Cx43 CT peptides identified by MS/MS as being cross-linked to αCT1. D) Tandem mass spectrum of a quintuply charged crosslinked peptide (m/z: 674.1) between Cx43 345-366 (a-chain) and αCT1 peptide through Cx43 K346 and E8 in αCT1 (b-chain). Only the b-and y-sequence specific ions are labeled. Arrow indicates ion (b a5 2+ ) consistent with cross-linkage between Cx43 CT lysine K346 and the glutamic acid (E) residue of αCT1 at position −1.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Blots of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT, GST-Cx43 CT QQ/KK in which the lysine (K) residues were mutated to neutral glutamines (Q), PKC-ε and αCT1 (at 5, 10 and 25 μM) and a scrambled αCT1 (M4 scr) variant at the same concentrations. Only αCT1 is seen to be covalently linked by EDC to Cx43 CT in a concentration-dependent manner.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of Cx43 and the secondary structure of Cx43 CT from amino acid residues Glycine252 (G252) through to Isoleucine 382 (I382). The depiction of secondary structure in 2A has been modified from a diagram originally provided by Sosinsky and co-workers . B) ZDOCK and C) Schrodinger molecular modeling software analysis of the structure of a proposed αCT1-Cx43 CT complex. The protonated structure of αCT1 peptide and Cx43 CT (PDB:1r5s), constrained by a salt-bridge interaction between K346 in the Cx43 CT and the glutamic acid (E) at position −1 of αCT1. The αCT1-Cx43 interaction shown represents that based on the lowest energy minimization score determined in the model. D) Schrodinger molecular modeling software, a 2D map of αCT1-Cx43 CT in anti-parallel orientation showing location of amino acids predicted to bond to each other and the type of bond that is predicted to occur.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Modification, Software

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: SPR was used to analyze interactions of biotin-αCT1 and biotin-αCT1 variant peptides, immobilized to streptavidin-coated chips, with the Cx43 CT (Cx43-CT: amino acids 255 to 382) and Cx43 CT-KK/QQ as analytes, respectively. The mean of three runs is plotted for each analyte concentration. The exposure of the sensor chip to the specific analyte is indicated by the gray area. Sensorgrams obtained for: A) Cx43 CT and biotin-αCT1. B) Cx43 CT-KK/QQ and biotin-αCT1. C) Cx43 CT and biotin-M1 AALAI. D) Cx43 CT-KK/QQ and biotin-M1 AALAI. E) Cx43 CT and biotin-M3 DDLAI. F) Cx43 CT-KK/QQ and biotin-M3 DDLAI.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Melt curves (top) and first derivative of melt curves (bottom) for ZO-1 PDZ2 at 500 μg/mL in combination αCT1 at concentrations of 25, 50 and 100 μM. B) Temperature maxima (Tm) from Boltzman curves from left-to-right of Cx43 CT (Cx43-CT: amino acids 255 to 382) alone, Cx43 CT in combination with αCT1, and the αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. αCT1, αCT1-I and αCT11 show similar abilities to destabilize (i.e., significantly decrease the Tm of) Cx43 CT. **p<0.01, *** p<0.002, N=6. C) Temperature maxima (Tm) from Boltzman curves from left-to-right of PDZ2 alone, and PDZ2 in combination with αCT1 and αCT1variants including αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. M3 DDLAI, αCT1, and αCT11 show similar abilities to stabilize (i.e., significantly increase the Tm of) PDZ2. **p<0.01, ***p<0.002, N=6

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques:

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with substrate (Cx43-CT: amino acids 255 to 382), but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-tagged αCT1, biotin-tagged αCT1 mutant peptides with alanine substitutions (M1 AALAI, M2 AALEI, M3 DDLAI) and biotin-tagged M4 scrambled. Peptides are at 20 μM. B) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with Cx43 CT substrate, but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-αCT1, biotin-αCT1-I or biotin-αCT11 (RPRPDDLEI with no antennapedia sequence at peptide NT) and biotin-M4 scrambled peptide. Peptides are at 20 μM. C) Chart showing that the ability of unmodified αCT1 and the Cx43 CT interaction-competent peptides biotin-αCT1-I or biotin-αCT11 to induce S368 phosphorylation was 3-5 fold greater than that of non-Cx43 CT interacting peptides. * p<0.05, ** p<0.01, *** p<0.002, N=5 αCT1 and M4, other peptides N=3.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff ischemia-reperfusion (I/R) injury protocols were performed on adult mouse hearts instrumented to monitor LV contractility (protocol in ). LV Systolic responses are shown in 7A-C : (A) Plots of left ventricular (LV) systolic developed pressure against balloon volume ; (B) LV maximal rate of tension development (+dP/dt) against balloon volume; (C) Maximal systolic elastance (E max ) – i.e., the slope from (A) ; (D) Plots of LV end diastolic pressure (EDP) against balloon volume; (E) Maximal rate of relaxation (-dP/dt) against balloon volume; (F) Stiffness, the reciprocal of the slope from (D) ; ( G) Percentage of LV contractile function recovery post-ischemia relative to baseline level. Data shown are mean ± S.E. N=4-8. *p<0.05, ***p<0.001, N=4-8 hearts/group. H) Blots of Cx43-pS368 (top) and total Cx43 (bottom) of LV samples infused with peptide for 20 minutes according to the protocol in . For hearts used in Western blots, the protocol did not proceed to the ischemia and reperfusion phases, being terminated after the peptide infusion step. Only those peptides competent to interact with Cx43 CT increase pS368 levels relative to total Cx43 above vehicle control.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff I/R protocols were performed on adult mouse hearts instrumented to monitor LV contractility. Protocol in , except that a 20-minute peptide infusion was begun after ischemic injury at the initiation of reperfusion. (A) Plots of left ventricular (LV) developed pressure against balloon volume; (B) Maximal systolic elastance (E max ), the slope from (A); (C) Maximal rate of tension development (+dP/dt) against balloon volume; (D) Plots of end diastolic pressure (EDP) against balloon volume; (E) Stiffness, the reciprocal of the slope from (D); (F) Maximal rate of relaxation (-dP/dt) against balloon volume. * p<0.05, *** p<0.001, N=4-8. G) Laser scanning confocal microscopic fields from sections of Vehicle control, αCT1, and αCT11 group hearts stained for Cx43 (green), nuclei (DAPI-blue), and Alexa647-conjugated streptavidin (red). H) Average intensities of biotinylated peptide (indicated by streptavidin Alexa647 fluorescence intensity level relative to background) in Vehicle control, αCT1, and αCT11 groups. ** p<0.05; not significant (ns) N=5 hearts/group. Scale bar = 5 μm.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative western blots suggest that neither total connexin43 (Cx43) nor Cx43 phosphorylated at serine-368 (pCx43-S368) significantly change in response to acute exposure to osmotic agents. Lane 7 contains a positive control using hearts exposed to 60 min of room temperature ischemia, a condition shown to upregulate Cx43 pS368. B. Summary data plotted with mean and standard deviation error bars do not reveal significant differences in either total or pCx43-S368 for all conditions (N=3 hearts per condition. p=n.s. via ordinary one-way ANOVA applying Dunnett’s correction). C. Representative fluorescent images of calcein-AM fluorescence in control cultures (N=13 plates, n=220 cells) and cultures exposed to mannitol (N=7 plates, n=116 cells), albumin (N=4 plates, n=77 cells), Dextran 70kDa (N=4 plates, n=102 cells), Dextran 2MDa (N=4 plates, n=95 cells), and carbenoxolone (CBX, N=6 plates, n=110 cells). Measurements were made before bleaching (Pre-bleach), just after bleaching (t=0s), halfway through and at the end of the recovery period (t=150 and 300s, respectively). D. Fluorescence recovery after photobleaching (FRAP) plot of bleached cells during 300s post bleaching. Solid lines represent averages at each point, and shaded areas represent the standard error of the mean. E. Normalized recovery demonstrates that only CBX significantly decreased FRAP relative to control. *p<0.05, nested ANOVA applying Dunnett’s correction with culture plate nested under treatment type.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Western Blot, Positive Control, Standard Deviation, Fluorescence, Control

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative images of ventricular tissue slices perfused with 0.5, 3, and 10kDa fluorescent dyes. Panels demonstrate extracellular diffusion; Cx43 signal is used to identify the intercalated disc (ID), and nuclei are shown alone and with overlay of all signals. B. A region of interest is identified around the Cx43 ID signal and intensity of signal is averaged to identify the peak of the Cx43 signal. The same spatial averaging with 0.5, 3, and 10kDa signal demonstrates a dip in 3 and 10kDa signal with a peak in the 0.5kDa dye demonstrating the 0.5kDa dye permeates the ID, but the other two dyes do not. C. The intensity of the dye fluorescent signal at the peak of the Cx43 signal is normalized to the intensity of signal beyond the ID to reveal that 3 (n=3 images) and 10kDa (n=4 images) fluorescent signal are significantly lower than 0.5kDa (n=7 images) signal intensity. *p<0.05 relative to 0.5kDa, one way ANOVA with Dunnett’s correction.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Diffusion-based Assay

Journal: European Heart Journal

Article Title: Left bundle branch vs biventricular pacing: mechanistic insights from a canine model

doi: 10.1093/eurheartj/ehaf1093

Figure Lengend Snippet: Molecular targets in cardiac remodeling by BiVP and LBBP. Western blotting results of BNP, TGF-β, KCNIP2, SERCA2a, Cx43, ANKRD1 protein expression (A, C, E) in septum and LV lateral walls from NC, DHF, and BiVP and LBBP groups, and relative protein expression (B, D, F). Represented images and statistical data (G) of cardiac fibrosis detected using Masson's staining. † P < .05 between septal and lateral walls. Abbreviations as in

Article Snippet: The levels of several key proteins were examined using western blot, including ANKRD1 (Proteintech, 11427-1-AP), BNP (Abclonal, A23996), Cx43 (Proteintech, 26980-1-AP), GSNOR (Proteintech, 66193-1-Ig), GLUD1 (Proteintech, 14299-1-AP), KCNIP2 (Abclonal, A7100), SERCA2a (Abclonal, A0098), TGF-β (Proteintech, 81746-2-RR), and GAPDH (Proteintech, 10494-1-AP).

Techniques: Western Blot, Expressing, Staining

Journal: European Heart Journal

Article Title: Left bundle branch vs biventricular pacing: mechanistic insights from a canine model

doi: 10.1093/eurheartj/ehaf1093

Figure Lengend Snippet: Overview of the key findings. This study compares the treatment efficacy of LBBP and BiVP, and relating electrocardiographic, echocardiographic and cellular (reverse) remodeling in a canine DHF model. The results show that LBBP restores electrical synchrony (QRSd) to a similar extent as BiVP, while leads to larger LV-GLS improvement than BiVP. Both LBBP and BiVP reverse the protein level of KCNIP2, SERCA2a and Cx43. Moreover, LBBP reverses abnormalities in cytoskeleton-associated proteins, TGF-β signaling, and SERCA2a expression more than BiVP, as well as enhances myocardial energy metabolism via more efficient TCA cycling and ATP production. ANKRD1, ankyrin repeat domain 1; ATP, adenosine triphosphate; BiVP, biventricular pacing; BNP, B-type natriuretic peptide; Cx43, connexin 43; DHF dyssynchronous heart failure; ECG, electrocardiogram; echo, echocardiogram; GLUD1, glutamate dehydrogenase 1; GSNOR S-nitrosoglutathione reductase; KCNIP2, potassium voltage-gated channel interacting protein 2; LBBP, left bundle branch pacing; LV-GLS, left ventricular global longitudinal strain; NC, normal control; SERCA2a, sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a; TCA, tricarboxylic acid; TGF-β, Transforming growth factor-β.

Article Snippet: The levels of several key proteins were examined using western blot, including ANKRD1 (Proteintech, 11427-1-AP), BNP (Abclonal, A23996), Cx43 (Proteintech, 26980-1-AP), GSNOR (Proteintech, 66193-1-Ig), GLUD1 (Proteintech, 14299-1-AP), KCNIP2 (Abclonal, A7100), SERCA2a (Abclonal, A0098), TGF-β (Proteintech, 81746-2-RR), and GAPDH (Proteintech, 10494-1-AP).

Techniques: Expressing, Control