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 + 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. (E) Resting membrane potential of WT and S3A cardiomyocytes. P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 vs. S3A vs. S3A + NO; P = 0.0001 S3A 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. (F) Representative current traces before and after application of 10 μM DEENO in a non-injected oocyte or an oocyte expressing Cx43 and Cx43S3A. Oocytes were clamped to −80 mV, and square pulses from −80 mV to +90 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to −80 mV. Intracellular injection of Gap19 (232 ng/μl) or a Cx43 CT antibody (2.5 ng/μl) reduces NO-induced Cx43 hemichannel currents. (G) Normalized currents were obtained from the ratio between recorded currents after and before DEENO treatment. The number in parentheses indicates biological replicate. We analyzed three to five oocytes per frog. P = 0.002 Cx43 vs. Cx43S3A; P = 0.0001 Cx43S3A vs. Cx43S3A + Gap19. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test. (H) Changes in resting membrane potential in the presence or absence of 10 μM DEENO. Cx43 hemichannel blockers restore normal resting membrane potential. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test.

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 + 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. (E) Resting membrane potential of WT and S3A cardiomyocytes. P = 0.0001 S3A vs. S3A + Iso; P = 0.0001 vs. S3A vs. S3A + NO; P = 0.0001 S3A 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. (F) Representative current traces before and after application of 10 μM DEENO in a non-injected oocyte or an oocyte expressing Cx43 and Cx43S3A. Oocytes were clamped to −80 mV, and square pulses from −80 mV to +90 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to −80 mV. Intracellular injection of Gap19 (232 ng/μl) or a Cx43 CT antibody (2.5 ng/μl) reduces NO-induced Cx43 hemichannel currents. (G) Normalized currents were obtained from the ratio between recorded currents after and before DEENO treatment. The number in parentheses indicates biological replicate. We analyzed three to five oocytes per frog. P = 0.002 Cx43 vs. Cx43S3A; P = 0.0001 Cx43S3A vs. Cx43S3A + Gap19. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test. (H) Changes in resting membrane potential in the presence or absence of 10 μM DEENO. Cx43 hemichannel blockers restore normal resting membrane potential. Comparisons between groups were made using two-way ANOVA plus Tukey’s post-hoc test.

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