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. 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. P = 0.006 WT vs. S3A; P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19 and S3A + Iso + abCx43. (E) Resting membrane potential of WT and S3A cardiomyocytes. The number in parentheses indicates the n total mice value. We analyzed three to five cells per mouse. P = 0.0001 WT vs. WT + Iso; P = 0.0015 WT vs. S3A; P = 0.0001 WT + Iso vs. S3A + Iso; P = 0.0001 S3A vs. S3A + Iso. Comparisons between groups were made using nested-ANOVA plus Tukey’s post-hoc test. (F) Evaluation of Cx43 hemichannel activity in the whole heart via ethidium uptake. Isolated hearts were perfused with buffer containing 5 μM ethidium after vehicle or Iso (5 mg/kg, IP). The number in parentheses indicates biological replicate. P = 0.0001 WT vs. WT + Iso; P = 0.0001 WT + Iso vs. S3A + Iso; P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test.

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. 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. P = 0.006 WT vs. S3A; P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19 and S3A + Iso + abCx43. (E) Resting membrane potential of WT and S3A cardiomyocytes. The number in parentheses indicates the n total mice value. We analyzed three to five cells per mouse. P = 0.0001 WT vs. WT + Iso; P = 0.0015 WT vs. S3A; P = 0.0001 WT + Iso vs. S3A + Iso; P = 0.0001 S3A vs. S3A + Iso. Comparisons between groups were made using nested-ANOVA plus Tukey’s post-hoc test. (F) Evaluation of Cx43 hemichannel activity in the whole heart via ethidium uptake. Isolated hearts were perfused with buffer containing 5 μM ethidium after vehicle or Iso (5 mg/kg, IP). The number in parentheses indicates biological replicate. P = 0.0001 WT vs. WT + Iso; P = 0.0001 WT + Iso vs. S3A + Iso; P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 S3A + Iso vs. S3A + Iso + Gap19. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test.

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