<i>In vivo</i> effect of NAC on gap junctional communication.

<p>Ventricular remodeling after MI was associated with marked decreased gap junctional communication, which was significantly increased after NAC administration. LY, Lucifer yellow; RD, rhodamine-conjugated dextran; Veh, vehicle. *p<0.05 compared with sham and NAC-treated group; †p<0.05 compared with sham.</p

cAMP-regulated PKA and Epac augment NAC-induced Cx43 levels in Experiment 2.

<p>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.</p

Western blot analysis of Cx43 and GSK-3β.

<p>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.</p

Model for the NAC signaling leads to the increase of Cx43 expression.

<p>The adenylate cyclase/cAMP signaling activates both PKA and Epac pathways resulting in phosphorylation of GSK-3β to act as a repressor for Cx43 gene transcription. Inhibition of these signaling pathways by their respective inhibitors is indicated by the <i>vertical lines</i> in reference to the direction of the <i>arrows</i>.</p

Role of GSK-3β in cAMP-dependent Cx43 levels in Experiment 3.

<p>In a rat isolated heart model, effect of GSK-3β was assessed on gap junctional communication and Western blot using antibodies against total Cx43, and phospho- and total-GSK-3β. Significantly increased Cx43 amount, P1/P0 ratio, and gap junctional communication are noted in groups treated with LiCl, LiCl+N6Bz, and LiCl+8-CPT compared with vehicle. 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 = 5 per group). LY, Lucifer yellow; P0, nonphosphorylated Cx43; P1, phosphorylated Cx43; RD, rhodamine-conjugated dextran. *p<0.01 compared with groups treated with vehicle, N6Bz, and 8-CPT; †p<0.05 compared with vehicle.</p

Representative immunofluorescent staining for Cx43.

<p>In sham, Cx43 forms clusters of punctate immunofluorescence domains confined to well-organized intercalated discs running across the longitudinal axis (magnification 400×). In contrast, infarction markedly decreases Cx43 proteins from the border zone. After administering NAC, Cx43 signals are increased. Bar = 50 µm. The proportion of the total cell area occupied by Cx43 immunoreactive signal at the border zone. Each column and bar represents mean ± SEM. *p<0.05 compared with sham and NAC-treated group; †p<0.05 compared with sham.</p

Cardiac morphology, hemodynamics, and cAMP levels at the end of study.

<p>Values are mean ± SEM. BW, body weight; HR, heart rate; LungW, lung weight; LVEDP, left ventricular end-diastolic pressure; LVESP, left ventricular end-systolic pressure; LVW, left ventricular weight; RVW, right ventricular weight.</p>*<p>p<0.05 compared with sham;</p>†<p>p<0.05 compared with the vehicle-treated group.</p

Gender-specific stratified analysis for CAS of hs-CRP tertiles, and diabetes mellitus or hypertension.

<p>Data are presented as multivariate-adjusted odds ratio (95% confidence interval), with adjusted variables including age, body mass index, smoking, diabetes mellitus, hypertension, left ventricular ejection fraction, cholesterol, hemoglobin, hematocrit and platelet other than the stratified variable <i>per se.</i> CAS, coronary artery spasm; hs-CRP: high-sensitivity C-reactive protein.</p

Univariate and multivariate Cox regression analysis for major adverse cardiovascular events and coronary events.

<p>CI, confidence interval; hs-CRP: high-sensitivity C-reactive protein.</p

Baseline characteristics.

<p>Data are presented as mean± standard deviation unless mentioned otherwise. A, before angiography; CAS, coronary artery spasm; D, at discharge; hs-CRP, high-sensitivity C-reactive protein.</p>*<p>hs-CRP samples were collected in a subset of 555 patients, with 223 and 332 in the control and CAS groups, respectively. Log-transformed values were used in analyses.</p