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Both PKA and Epac pathways mediate N-acetylcysteine-induced Connexin43 preservation in rats with myocardial infarction.

Lee TM, Lin SZ, Chang NC - PLoS ONE (2013)

Bottom Line: Cardiac remodeling was shown to be associated with reduced gap junction expression after myocardial infarction.In an ex vivo study, enhanced connexin43 levels afforded by N-acetylcysteine were partially blocked by either H-89 (a PKA inhibitor) or brefeldin A (an Epac-signaling inhibitor) and completely blocked when H-89 and brefeldin A were given in combination.Addition of either the PKA specific activator N6Bz or Epac specific activator 8-CPT did not have additional increased connexin43 levels compared with rats treated with lithium chloride alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Cardiology Section, Tainan Municipal An-Nan Hospital-China Medical University, Tainan, Taiwan ; Department of Medicine, China Medical University, Taichung, Taiwan ; Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

ABSTRACT
Cardiac remodeling was shown to be associated with reduced gap junction expression after myocardial infarction. A reduction in gap junctional proteins between myocytes may trigger ventricular arrhythmia. Therefore, we investigated whether N-acetylcysteine exerted antiarrhythmic effect by preserving connexin43 expression in postinfarcted rats, focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). Male Wistar rats after ligating coronary artery were randomized to either vehicle, or N-acetylcysteine for 4 weeks starting 24 hours after operation. Infarct size was similar between two groups. Compared with vehicle, cAMP levels were increased by N-acetylcysteine treatment after infarction. Myocardial connexin43 expression was significantly decreased in vehicle-treated infarcted rats compared with sham operated rats. Attenuated connexin43 expression and function were blunted after administering N-acetylcysteine, assessed by immunofluorescent analysis, dye coupling, Western blotting, and real-time quantitative RT-PCR of connexin43. Arrhythmic scores during programmed stimulation in the N-acetylcysteine-treated rats were significantly lower than those treated with vehicle. In an ex vivo study, enhanced connexin43 levels afforded by N-acetylcysteine were partially blocked by either H-89 (a PKA inhibitor) or brefeldin A (an Epac-signaling inhibitor) and completely blocked when H-89 and brefeldin A were given in combination. Addition of either the PKA specific activator N6Bz or Epac specific activator 8-CPT did not have additional increased connexin43 levels compared with rats treated with lithium chloride alone. These findings suggest that N-acetylcysteine protects ventricular arrhythmias by attenuating reduced connexin43 expression and function via both PKA- and Epac-dependent pathways, which converge through the inactivation of glycogen synthase kinase-3β.

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cAMP-regulated PKA and Epac augment NAC-induced Cx43 levels in Experiment 2.In a rat isolated heart model, effect of PKA and Epac was assessed on gap junctional communication and Western blot using antibody against Cx43 and GSK-3β. Significantly decreased Cx43 amount, P1/P0 ratio, and gap junctional communication are noted in the groups treated with SQ-22536, H-89 and brefeldin A compared with NAC alone. Compared with vehicle (Veh), NAC administration showed significantly decreased p-GSK-3β, which can be partially reversed after adding either H-89 or brefeldin A. SQ-22536 significantly reduced augmentation of NAC-induced Cx43 levels to a much greater extent than either H-89 or brefeldin A alone. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Each point is an average of 3 separate experiments (n = 10 per group). LY, Lucifer yellow; P0, nonphosphorylated Cx43; P1, phosphorylated Cx43; RD, rhodamine-conjugated dextran. *p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, NAC+H-89, NAC+brefeldin A, and NAC+H-89+brefeldin A; †p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, and NAC+H-89+brefeldin A.
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pone-0071878-g005: cAMP-regulated PKA and Epac augment NAC-induced Cx43 levels in Experiment 2.In a rat isolated heart model, effect of PKA and Epac was assessed on gap junctional communication and Western blot using antibody against Cx43 and GSK-3β. Significantly decreased Cx43 amount, P1/P0 ratio, and gap junctional communication are noted in the groups treated with SQ-22536, H-89 and brefeldin A compared with NAC alone. Compared with vehicle (Veh), NAC administration showed significantly decreased p-GSK-3β, which can be partially reversed after adding either H-89 or brefeldin A. SQ-22536 significantly reduced augmentation of NAC-induced Cx43 levels to a much greater extent than either H-89 or brefeldin A alone. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Each point is an average of 3 separate experiments (n = 10 per group). LY, Lucifer yellow; P0, nonphosphorylated Cx43; P1, phosphorylated Cx43; RD, rhodamine-conjugated dextran. *p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, NAC+H-89, NAC+brefeldin A, and NAC+H-89+brefeldin A; †p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, and NAC+H-89+brefeldin A.

Mentions: Western blot findings showed that the Cx43 protein levels were augmented by NAC compared with vehicle in a rat isolated post-infarcted heart model (Figure 5). The adenylate cyclase inhibitor SQ-22526 inhibited the NAC-induced Cx43 levels, implying the important role of cAMP in increasing Cx43 levels. These results are in agreement with previous reports [32] and validated the assays we used in this study.


Both PKA and Epac pathways mediate N-acetylcysteine-induced Connexin43 preservation in rats with myocardial infarction.

Lee TM, Lin SZ, Chang NC - PLoS ONE (2013)

cAMP-regulated PKA and Epac augment NAC-induced Cx43 levels in Experiment 2.In a rat isolated heart model, effect of PKA and Epac was assessed on gap junctional communication and Western blot using antibody against Cx43 and GSK-3β. Significantly decreased Cx43 amount, P1/P0 ratio, and gap junctional communication are noted in the groups treated with SQ-22536, H-89 and brefeldin A compared with NAC alone. Compared with vehicle (Veh), NAC administration showed significantly decreased p-GSK-3β, which can be partially reversed after adding either H-89 or brefeldin A. SQ-22536 significantly reduced augmentation of NAC-induced Cx43 levels to a much greater extent than either H-89 or brefeldin A alone. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Each point is an average of 3 separate experiments (n = 10 per group). LY, Lucifer yellow; P0, nonphosphorylated Cx43; P1, phosphorylated Cx43; RD, rhodamine-conjugated dextran. *p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, NAC+H-89, NAC+brefeldin A, and NAC+H-89+brefeldin A; †p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, and NAC+H-89+brefeldin A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756050&req=5

pone-0071878-g005: cAMP-regulated PKA and Epac augment NAC-induced Cx43 levels in Experiment 2.In a rat isolated heart model, effect of PKA and Epac was assessed on gap junctional communication and Western blot using antibody against Cx43 and GSK-3β. Significantly decreased Cx43 amount, P1/P0 ratio, and gap junctional communication are noted in the groups treated with SQ-22536, H-89 and brefeldin A compared with NAC alone. Compared with vehicle (Veh), NAC administration showed significantly decreased p-GSK-3β, which can be partially reversed after adding either H-89 or brefeldin A. SQ-22536 significantly reduced augmentation of NAC-induced Cx43 levels to a much greater extent than either H-89 or brefeldin A alone. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Each point is an average of 3 separate experiments (n = 10 per group). LY, Lucifer yellow; P0, nonphosphorylated Cx43; P1, phosphorylated Cx43; RD, rhodamine-conjugated dextran. *p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, NAC+H-89, NAC+brefeldin A, and NAC+H-89+brefeldin A; †p<0.05 compared with groups treated with vehicle, NAC+SQ-22536, and NAC+H-89+brefeldin A.
Mentions: Western blot findings showed that the Cx43 protein levels were augmented by NAC compared with vehicle in a rat isolated post-infarcted heart model (Figure 5). The adenylate cyclase inhibitor SQ-22526 inhibited the NAC-induced Cx43 levels, implying the important role of cAMP in increasing Cx43 levels. These results are in agreement with previous reports [32] and validated the assays we used in this study.

Bottom Line: Cardiac remodeling was shown to be associated with reduced gap junction expression after myocardial infarction.In an ex vivo study, enhanced connexin43 levels afforded by N-acetylcysteine were partially blocked by either H-89 (a PKA inhibitor) or brefeldin A (an Epac-signaling inhibitor) and completely blocked when H-89 and brefeldin A were given in combination.Addition of either the PKA specific activator N6Bz or Epac specific activator 8-CPT did not have additional increased connexin43 levels compared with rats treated with lithium chloride alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Cardiology Section, Tainan Municipal An-Nan Hospital-China Medical University, Tainan, Taiwan ; Department of Medicine, China Medical University, Taichung, Taiwan ; Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

ABSTRACT
Cardiac remodeling was shown to be associated with reduced gap junction expression after myocardial infarction. A reduction in gap junctional proteins between myocytes may trigger ventricular arrhythmia. Therefore, we investigated whether N-acetylcysteine exerted antiarrhythmic effect by preserving connexin43 expression in postinfarcted rats, focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). Male Wistar rats after ligating coronary artery were randomized to either vehicle, or N-acetylcysteine for 4 weeks starting 24 hours after operation. Infarct size was similar between two groups. Compared with vehicle, cAMP levels were increased by N-acetylcysteine treatment after infarction. Myocardial connexin43 expression was significantly decreased in vehicle-treated infarcted rats compared with sham operated rats. Attenuated connexin43 expression and function were blunted after administering N-acetylcysteine, assessed by immunofluorescent analysis, dye coupling, Western blotting, and real-time quantitative RT-PCR of connexin43. Arrhythmic scores during programmed stimulation in the N-acetylcysteine-treated rats were significantly lower than those treated with vehicle. In an ex vivo study, enhanced connexin43 levels afforded by N-acetylcysteine were partially blocked by either H-89 (a PKA inhibitor) or brefeldin A (an Epac-signaling inhibitor) and completely blocked when H-89 and brefeldin A were given in combination. Addition of either the PKA specific activator N6Bz or Epac specific activator 8-CPT did not have additional increased connexin43 levels compared with rats treated with lithium chloride alone. These findings suggest that N-acetylcysteine protects ventricular arrhythmias by attenuating reduced connexin43 expression and function via both PKA- and Epac-dependent pathways, which converge through the inactivation of glycogen synthase kinase-3β.

Show MeSH
Related in: MedlinePlus