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. 2013 Jan 24;14(2):2242-57.
doi: 10.3390/ijms14022242.

Advanced Glycation End Product (AGE)-AGE Receptor (RAGE) System Upregulated Connexin43 Expression in Rat Cardiomyocytes via PKC and Erk MAPK Pathways

Lu Yu  1 , Yanbo Zhao, Shengjie Xu, Fang Ding, Chongying Jin, Guosheng Fu, Shaoxiang Weng
Affiliations

Advanced Glycation End Product (AGE)-AGE Receptor (RAGE) System Upregulated Connexin43 Expression in Rat Cardiomyocytes via PKC and Erk MAPK Pathways

Lu Yu et al. Int J Mol Sci. .

Abstract

The remodeling of cardiac gap junction contributes to the arrhythmias in a diabetic heart. We previously reported that high glucose reduced Cx43 protein level in neonatal rat cardiomyocytes. But, the effect and mechanisms of advanced glycation end product (AGE) on Cx43 expression still remain unclear. In this study, we measured the AGE receptor (RAGE) and Cx43 expression by immunohistochemisty in AGE-infused Sprague-Dawley (SD) rats. In vitro, the Cx43 and RAGE levels were detected in AGE-treated cardiomyocytes by Western blot and real-time RT-PCR. The function of cells coupling was measured by Scrap loading dye transfer assay. Our results showed that the AGE-infused rat hearts exhibited increased cardiac RAGE and Cx43, as well as Cx43 redistribution. In cultured cardiomyocytes, AGE elevated RAGE expression in a time- and dose-dependent manner. Cx43 protein and mRNA levels were upregulated by AGE (200 mg/L, 24 h), but the gap junction function was not enhanced. RAGE-targeted knock-down or the addition of PKC, and Erk inhibitors abolished the effect of AGE on Cx43. Therefore, AGE-RAGE system might elevate Cx43 expression in rat cardiomyocytes by activating PKC and Erk MAPK pathways, and it also enhanced Cx43 redistribution in vivo, which might contribute to the arrhythmias in diabetes.

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Figures

Figure 1
Figure 1
The advanced glycation end product (AGE)-AGE receptor (RAGE) and Cx43 staining in rat heart tissue. (A) The RAGE and Cx43 expression detected by immuohistochemisty; (B) Quantity analysis of staining assessed by ImageJ software. bovine serum albumin (BSA): BSA-infused rat (40 mg/kg/d); AGE: AGE-infused rat (40 mg/kg/d). Black arrow: RAGE; White arrow: Cx43. Bar: 100 μM. * p < 0.05 vs. control.
Figure 2
Figure 2
Effect of AGE on the cell viability determined by MTT assay. (A) Cells were treated with AGE at the concentration of 50, 100 and 200 mg/L for 24 h; (B) Cells were treated with AGE at 200 mg/L for 6, 12, 24 and 48 h respectively. n = 5 wells in each individual experiment. * p < 0.05 vs. control; Data are mean ± SD.
Figure 3
Figure 3
The effect of AGE on RAGE expression in cardiomyocytes. (A) AGE upregulated the RAGE protein expression in a dose-dependent manner and peaked at 200 mg/L; (B) AGE increased the RAGE protein expression in a time-dependent manner and peaked at 24 h; (C) RAGE mRNA level was upregulated by AGE (200 mg/L) treatment for 24 h. * p < 0.05 vs. control, BSA (200 mg/L); # p < 0.05 vs. AGE (50 mg/L, 100 mg/L) or AGE (6h, 12h, 48 h); Data are mean ± SD.
Figure 4
Figure 4
The effect of AGE on Cx43 expression and gap junctional intercellular communication (GJIC) function. (A) Cx43 protein (P0, P1, P2) expression was upregulated by AGE (200 mg/L) treatment for 24 h; (B) Cx43 mRNA level was upregulated by AGE (200 mg/L) treatment for 24 h. (C) and (D) showed effect of AGE on the GJIC function assessed by Scrape loading dye transfer assay; (E) The quantity analysis of dye transfer distance in each group. * p < 0.05 vs. control, BSA (200 mg/L); White arrow: scrape line; Data are mean ± SD.
Figure 5
Figure 5
The effect of AGE on Cx43 expression in cardiomyocytes with RAGE knocked down. (A) The identification of RAGE expression knocked down with small interfering (siRNA) assessed by Western blot and real-time polymerase chain reaction (PCR); (B) The effect of AGE on Cx43 protein in cardiomyocytes with or without RAGE knocked down. * p < 0.05 vs. Scr (Scrambled siRNA); siRNA: RAGE-targeted siRNA. Data are mean ± SD.
Figure 6
Figure 6
AGE elevated time-dependent phosphorylation levels of JNK (A); Erk (B) and P38 MAPK (C) in cardiomyocytes determined by Western blot. (D) Western blot analysis of Cx43 expression in AGE-treated cardiomyocytes with the presence of MAPK inhibitors. * p < 0.05 vs. control; # p < 0.05 vs. AGE; Data are mean ± SD.
Figure 7
Figure 7
The involvement of the PKC pathway in the effect of AGE on Cx43 expression. (A) AGE activated the time-dependent phosphorylation of PKCα/β2 in cardiomyocytes assessed by Western blot; (B) Cx43 protein expression in cells treated by AGE for 24 h with the presence of PKC inhibitor GF109203X (GF, 10 μM). * p < 0.05 vs. control; Data are mean ± SD.
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