Remodeled connexin 43 hemichannels alter cardiac excitability and promote arrhythmias

Figure 1.. Cx43 hemichannels are lateralized in cardiomyocytes of S3A mice. (A) Representative confocal immunofluorescence images of cardiac intercalated discs and lateral regions of cardiomyocytes stained with Cx43 (green) and N-cadherin (red). Yellow and white arrows indicate the intercalated disc and lateral side, respectively. Right: Quantification of Cx43/N-cadherin colocalization in confocal immunofluorescence images. All data points were normalized to the WT group mean. Between three and five images containing 15–20 IDs were analyzed per heart. Each dot represents the mean value for each biological replicate. Comparisons between groups were made using Student’s t test, P = 0.0001 vs. WT. The number in parentheses indicates the n total mice value. (B) Western blot analysis (left) and quantification (right) of Cx43 from biotin-perfused hearts (biotinylation). The bottom row represents Cx43-immunoblotted samples from heart lysates prior to pulldown (total Cx43). Biotinylated Cx43 levels were expressed as fold change relative to total Cx43 protein levels per sample. The number in parentheses indicates the n total mice value. Comparisons between groups were made using Student’s t test, P…

Figure 2.. β-adrenergic cardiac stress promotes arrhythmogenic phenotypes in S3A mice, which are prevented by blocking Cx43 hemichannels. (A) Representative ECG traces obtained from 4–6-mo-old mice using an in vivo telemetry system in WT, S3A, and S3A mice treated with Gap19 via retroorbital injection (10 μg/kg). Scale bar is 100 ms for all traces. Black arrows indicate PVC arrhythmia events. (B) Arrhythmia scores 1-h post-Iso challenge based on a predetermined scale where 0 = no arrhythmias, 1 = single PVCs, 2 = double PVCs, 3 = triple PVCs or non-sustained VT, 4 = sustained VT or AV block, 5 = death. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test, P = 0.0001 WT vs. S3A; P = 0.0001 S3A vs. S3A + Gap19. The number in parentheses indicates biological replicates. (C) Quantification of arrhythmogenic events (including PVC, double PVC, VT, or AV block) during the 24 h post-Iso challenge in WT, S3A, and S3A mice treated with Gap19 via retroorbital injection (10 μg/kg). P = 0.0001 WT vs. S3A; P = 0.0001 S3A vs. S3A + Gap19; P = 0.00703 WT vs. S3A + Gap19. The number in parentheses indicates biological replicates. Comparisons between groups were made using…

Figure 3.. Isoproterenol induces TA in S3A cardiomyocytes via the opening of Cx43 hemichannels. (A) Representative AP traces of WT and S3A isolated cardiomyocytes. Cells were stimulated with 1 µM Iso (green) in the absence or presence of Cx43 blockers contained inside the pipette: Gap19 (232 ng/μl) and Cx43 antibody (abCx43; 2.5 ng/μl). Black arrows indicate electrical stimulation pulse. Red arrows indicate TAs. Red asterisks display DADs. (B) Quantification of DADs observed in A. P = 0.0001 WT + Iso vs. S3A; P = 0.0008 WT + Iso vs. .S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19 and S3A + Iso + abCx43. Comparisons between groups were made using nested-ANOVA plus Tukey’s post-hoc test. The number in parentheses indicates biological replicates. We analyzed three to five cells per mouse. (C) Quantification of DADs amplitude observed in A. P = 0.0001 WT vs. S3A; P = 0.002 S3A vs. S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19 and S3A + Iso + abCx43. Comparisons between groups were made using nested-ANOVA plus Tukey’s post-hoc test. The number in parentheses indicates biological replicates. We analyzed three to five cells per mouse. (D) Quantification of TA observed in A.…

Figure S1.. Injection of hyperpolarizing current is sufficient to regulate DAD and TA in isolated S3A cardiomyocytes. (A) Representative APs traces of S3A isolated cardiomyocytes in the absence or presence of 1 μM Iso. Hyperpolarizing currents (20–12 pA) were injected to maintain the resting membrane potential close to −68 mV (value observed in WT cardiomyocytes). Black arrows indicate electrical stimulation pulse. Red arrows indicate TAs. Red asterisks display DADs. (B) Quantification of DADs observed in 1 min. (C) Quantification of DADs amplitude. (D) Quantification of TAs detected in 1 min. The number in parentheses indicates biological replicates. Comparisons between groups were made using nested-ANOVA plus Tukey’s post-hoc test.

Figure 4.. S3A cardiomyocytes show an increased duration of [Ca2+]i transients compared to WT cardiomyocytes, which was prevented by Cx43 hemichannel blockade. (A and B) Representative line scan images from WT (top) and S3A (bottom) myocytes under field stimulation (1 Hz) in control conditions (A) and after incubation with Gap19 peptide (200 µM; B). The traces above each line scan represent the spatially averaged time curse of [Ca2+]i for each image. The number in parentheses indicates biological replicates. We analyzed three to five cells per mouse. (C and D) Amplitude quantification (C) and decay time 90% (ms) quantification (D). The number in parentheses indicates the n value. P = 0.0012 WT vs. S3A; P = 0.0067 S3A vs. S3A + Gap19. Comparisons between groups were made using nested-ANOVA. The number in parentheses indicates biological replicates. We analyzed three to five cells per mouse.

Figure 5.. Activation of β-adrenergic signaling decreases the duration of [Ca2+]i transients in S3A cardiomyocytes treated with Gap19. (A) Line scan images from representative WT (top) and S3A (bottom) cardiomyocytes under field stimulation (1 Hz) under control conditions (left) and after isoproterenol stimulation (right). (B) Line scan images from representative cardiomyocytes previously loaded with the Gap19 peptide (200 µM) before (left) and after (right) Iso stimulation (green). The traces above each line scan represent the spatially averaged time curse of [Ca2+]i for each image. The number in parentheses indicates biological replicates. (C and D) We analyzed three to five cells per mouse. C and D show scatter plots of the amplitude and time to 90% decay, respectively. P = 0.0203 WT vs. S3A; P = 0.0164 S3A vs. S3A + Iso + Gap19. The number in parentheses indicates the n value. Comparisons between groups were made using nested-ANOVA. The number in parentheses indicates biological replicate. We analyzed three to five cells per mice.

Figure 6.. Cx43 hemichannels are involved in APD prolongation in WT and S3A isolated cardiomyocytes. (A) Representative AP traces of WT and S3A isolated cardiomyocytes. Cells were stimulated with 1 µM Iso (green) in the absence or presence of Cx43 blockers contained inside the pipette: Gap19 (232 ng/μl) and Cx43 CT antibody (abCx43; 2.5 ng/μl). (B) Quantification of APD observed in A. P = 0.0001 WT vs. WT + Iso; P = 0.0001 WT + Iso vs. WT + Iso + abCx43, P = 0.0001 WT + Iso vs. S3A + Iso, P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19 and S3A + Iso + abCx43. Comparisons between groups were made using nested-ANOVA. The number in parentheses indicates biological replicate. We analyzed three to five cells per mouse.

Figure 7.. β-adrenergic cardiac stress evokes triggered activity in S3A cardiac cells via S-nitrosylation of lateralized Cx43 hemichannels. (A) Representative AP traces of S3A isolated cardiomyocytes. Cells were stimulated with 1 μM Iso in the presence of 100 μM L-NAME (NOS blocker) or 1 µM DEENO (NO donor). Arrow indicates electrical stimulation. Black arrows indicate electrical stimulation pulse. Red arrows indicate TAs. Red asterisks display DADs. (B) Quantification of DADs observed in A. P = 0.0001 S3A vs. S3A + L-NAME, P = 0.0001 S3A + Iso vs. S3A + ISO + L-NAME, P = 0.0195 S3A + L-NAME vs. S3A + L-NAME + Iso. Comparisons between groups were made using nested-ANOVA. The number in parentheses indicates biological replicates. We analyzed three to five cells per mouse. (C) Quantification of DADs amplitude observed in A. P = 0.0390 S3A vs. S3A + L-NAME, P = 0.0034 S3A + Iso vs. S3A + L-NAME, P = 0.0001 NO vs. S3A + L-NAME. Comparisons between groups were made using nested-ANOVA. The number in parentheses indicates biological replicates. We analyzed three to five cells per mouse. (D) Quantification of TA observed in A. P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso +…

Figure 8.. β-adrenergic cardiac stress evokes S-nitrosylation lateralized Cx43 proteins. (A) Representative images of analysis performed by PLA of the interaction between Cx43 and S-nitrosylation. Plasma membrane stained with wheat germ agglutinin (WGA) and S-nitrosylated Cx43 (Cx43-SNO) are shown in green and red, respectively. Scale bar: 50 μm. (B) Quantification of PLA dots signals observed in A. P = 0.015 WT vs. WT + Iso; P = 0.0001 WT + Iso vs. S3A + Iso; P = 0.0001 S3A vs. S3A + Iso. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test. The number in parentheses indicates biological replicate. (C) The relative percentage of Cx43-NO dots signal at the lateral side of cardiomyocytes detected in A. Note that S3A-Iso enhanced lateralized signal. P = 0.0001 WT vs. S3A + Iso; P = 0.0001 WT + Iso vs. S3A + Iso; P = 0.0001 S3A vs. S3A + Iso. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test. The number in parentheses indicates biological replicate. (D) Analysis of cell areas are detected in WT and S3A mice under a vehicle and upon Iso stimulation. (E) Top and middle gels were loaded with S-nitrosylated proteins pulled down using…

Figure S2.. S3A mice displayed arrhythmogenic behaviors during the dark cycle with and without Iso. (A) Quantification of arrhythmogenic events (including PVC, double PVC, VT, or AV block) from 4–6-mo-old mice during the dark and light cycle without Iso in WT, S3A, and S3A mice treated with Gap19 via retroorbital injection (10 μg/kg). The number in parentheses indicates the n value. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test. (B) Quantification of arrhythmogenic events (including PVC, double PVC, VT, or AV block) from 4–6-mo-old mice during the dark and light cycle upon Iso stimulation (5 mg/kg, IP) in WT, S3A, and S3A mice treated with Gap19 via retroorbital injection (10 μg/kg). The number in parentheses indicates biological replicate. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test. (C) Movements were detected using an in vivo telemetry system in WT, S3A, and S3A mice treated with Gap19 via retroorbital injection (10 μg/kg). Arrow indicates Iso stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test.