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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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Western blot analysis of Cx43 and GSK-3β.Ventricular remodeling after MI was associated with marked decreased amount of phosphorylated Cx43 (P1). Significantly increased Cx43 amount and P1/P0 ratio had taken place in the NAC-treated group compared with vehicle (Veh). However, a significantly decreased p-GSK-3β is noted in the NAC-treated group compared with vehicle. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Densitometric quantification of phosphorylation levels was expressed as the ratio of the density of phosphorylated band over total GSK-3β. Each point is an average of 3 separate experiments. P0, nonphosphorylated Cx43; P1, phosphorylated Cx43. *p<0.05 compared with sham and NAC-treated group; †p<0.05 compared with sham.
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pone-0071878-g002: Western blot analysis of Cx43 and GSK-3β.Ventricular remodeling after MI was associated with marked decreased amount of phosphorylated Cx43 (P1). Significantly increased Cx43 amount and P1/P0 ratio had taken place in the NAC-treated group compared with vehicle (Veh). However, a significantly decreased p-GSK-3β is noted in the NAC-treated group compared with vehicle. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Densitometric quantification of phosphorylation levels was expressed as the ratio of the density of phosphorylated band over total GSK-3β. Each point is an average of 3 separate experiments. P0, nonphosphorylated Cx43; P1, phosphorylated Cx43. *p<0.05 compared with sham and NAC-treated group; †p<0.05 compared with sham.

Mentions: Western blot showed that ventricular remodeling is associated with progressively decreased amount of phosphorylated- and total Cx43 at the border zone (Figure 2). In the NAC-treated group, total Cx43 amount was maintained at 68% of that in the sham group, a 45% higher than that in the vehicle-treated group. Furthermore, P1/P0 ratio was significantly increased in the NAC-treated groups compared with vehicle (3.4±0.3 vs. 1.5±0.3, p<0.001). Western blot data were consistent with immunohistochemical data analysis.


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)

Western blot analysis of Cx43 and GSK-3β.Ventricular remodeling after MI was associated with marked decreased amount of phosphorylated Cx43 (P1). Significantly increased Cx43 amount and P1/P0 ratio had taken place in the NAC-treated group compared with vehicle (Veh). However, a significantly decreased p-GSK-3β is noted in the NAC-treated group compared with vehicle. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Densitometric quantification of phosphorylation levels was expressed as the ratio of the density of phosphorylated band over total GSK-3β. Each point is an average of 3 separate experiments. P0, nonphosphorylated Cx43; P1, phosphorylated Cx43. *p<0.05 compared with sham and NAC-treated group; †p<0.05 compared with sham.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756050&req=5

pone-0071878-g002: Western blot analysis of Cx43 and GSK-3β.Ventricular remodeling after MI was associated with marked decreased amount of phosphorylated Cx43 (P1). Significantly increased Cx43 amount and P1/P0 ratio had taken place in the NAC-treated group compared with vehicle (Veh). However, a significantly decreased p-GSK-3β is noted in the NAC-treated group compared with vehicle. Relative abundance was obtained by normalizing the density of Cx43 protein against that of β-actin. Densitometric quantification of phosphorylation levels was expressed as the ratio of the density of phosphorylated band over total GSK-3β. Each point is an average of 3 separate experiments. P0, nonphosphorylated Cx43; P1, phosphorylated Cx43. *p<0.05 compared with sham and NAC-treated group; †p<0.05 compared with sham.
Mentions: Western blot showed that ventricular remodeling is associated with progressively decreased amount of phosphorylated- and total Cx43 at the border zone (Figure 2). In the NAC-treated group, total Cx43 amount was maintained at 68% of that in the sham group, a 45% higher than that in the vehicle-treated group. Furthermore, P1/P0 ratio was significantly increased in the NAC-treated groups compared with vehicle (3.4±0.3 vs. 1.5±0.3, p<0.001). Western blot data were consistent with immunohistochemical data analysis.

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