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Angiotensin II reduces cardiac AdipoR1 expression through AT1 receptor/ROS/ERK1/2/c-Myc pathway - PubMed

Angiotensin II reduces cardiac AdipoR1 expression through AT1 receptor/ROS/ERK1/2/c-Myc pathway

Li Li et al. PLoS One. 2013.

Abstract

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AngII infusion reduces plasma adiponectin levels, inhibits cardiac AdipoR1 expression, and decreases phosphorylation of AMPK and ACC.

(A) Plasma adiponectin levels in the control, AngII, and losartan groups were detected by ELISA. (B) Levels of AdipoR1 mRNA were analyzed by qRT-PCR, and β-actin was used as an internal control. (C) Myocardial extracts were immunoblotted with antibody specific for AdipoR1. Blots were reprobed with actin to confirm equal loading. (D) Representative immunostaining images and averaged bar graphs of AdipoR1 density. AdipoR1 is shown in brown and cell nuclei in blue. Scale bar represents 10 µm (6 fields in each sample were scanned and averaged). (E) Myocardial extracts were immunoblotted with antibody specific for p-AMPKα at Thr-172. Blots were reprobed with antibody for AMPK to confirm equal loading. (F) Myocardial extracts were immunoblotted with antibody specific for p-ACC at Ser-79. Blots were reprobed with antibody for ACC to confirm equal loading. Data represent mean ± SE. n = 6 in each group. **P<0.01 vs. control. # P<0.05, ## P<0.01 vs. AngII.

Figure 2
Figure 2. AngII decreases AdipoR1 expression in NRVMs. NRVMs were incubated with 0.1 μmol/L AngII for the indicated times, or treated with AngII at the indicated concentrations for 48 h.

(A, C) Levels of AdipoR1 mRNA were analyzed by qRT-PCR. (B, D) Expression of AdipoR1 protein was determined by Western blot analysis. (E) Representative immunofluorescence images and averaged bar graphs of AdipoR1 density in unstimulated control NRVMs and cells treated with 0.1 µmol/L AngII for 48 h. Green fluorescence signals represent AdipoR1 protein. Scale bar represents 20 µm (6 fields in each sample were scanned and averaged). (F) NRVMs were pretreated with 0.1 µmol/L AngII for 48 h and then stimulated with 1 µg/ml globular adiponectin for 10 min. AMPK phosphorylation in cell lysates was determined by Western blot analysis. Data represent mean ± SE of three independent experiments. **P<0.01 vs. control. ## P<0.01 vs. AngII.

Figure 3
Figure 3. The AngII-induced decrease in AdipoR1 expression is AT1 receptor-dependent.

NRVMs were pretreated with losartan (10 µmol/L) or PD123319 (1 µmol/L) for 1 h and then stimulated with 0.1 µmol/L AngII for 48 h, or incubated with CGP42112A (1 µmol/L) for 48 h. (A) Levels of AdipoR1 mRNA were analyzed by qRT-PCR, and β-actin was used as an internal control. (B) Western blot was performed to detect levels of AdipoR1 and actin protein expression. Data represent mean ± SE of three independent experiments. *P<0.05, **P<0.01 vs. control. # P<0.05 vs. AngII.

Figure 4
Figure 4. The AngII-induced decrease in AdipoR1 expression involves ERK1/2 activation and ROS production.

(A, B) NRVMs were pretreated with SB202190 (5 µmol/L), PD98059 (10 µmol/L), or SP600125 (10 µmol/L) for 1 h, then stimulated with 0.1 µmol/L AngII for 48 h. Expression of AdipoR1 mRNA and protein was analyzed by qRT-PCR and Western blot respectively. (C) Representative fluorescence images and histogram of relative fluorescence density of ROS. NRVMs were pretreated with NAC (10 mmol/L), apocynin (100 µmol/L), rotenone (10 µmol/L), or allopurinol (100 µmol/L) for 1 h, followed by DCF-DA incubation for 30 min, and then stimulated with 0.1 µmol/L AngII for 30 min. DCF fluorescence was visualized using confocal microscopy. The green color represents ROS and the fluorescence density was normalized to cell count. Scale bar represents 20 µm (6 fields in each sample were scanned and averaged). (D, E) NRVMs were pretreated with NAC (10 mmol/L), apocynin (100 µmol/L), rotenone (10 µmol/L), or allopurinol (100 µmol/L) for 1 h and then stimulated with 0.1 µmol/L AngII for 30 min (D) or 48 h (E). Western blot was performed to detect levels of p-ERK1/2 (D) or AdipoR1 (E). Blots were reprobed with antibody for ERK1/2 (D) or actin (E) to confirm equal loading. (F, G) NRVMs were pretreated with 10 µmol/L gp91 ds-tat or scrambled gp91 ds-tat for 1 h and then stimulated with 0.1 µmol/L AngII for 30 min (F) or 48 h (G). Western blot was performed to detect levels of p-ERK1/2 (F) or AdipoR1 (G). Data represent mean ± SE of three independent experiments. *P<0.05, **P<0.01 vs. control. # P<0.05, ## P<0.01 vs. AngII.

Figure 5
Figure 5. AngII promotes binding of c-Myc to the AdipoR1 promoter region and it requires ERK1/2 involvement.

(A) NRVMs were stimulated with 0.1 µmol/L AngII for 48 h. Sheared chromatin prepared from cardiomyocytes was immunoprecipitated with the indicated antibodies (control IgG, anti-c-Myc, anti-STAT5 or anti-NF-κB antibodies). The immunoprecipitated DNA was used as a template for PCR using the indicated primer sets. (B) NRVMs were pretreated with 10 µmol/L PD98059 for 1 h, then stimulated with 0.1 µmol/L AngII for 48 h. Chromatin fragments were immunoprecipitated with anti-c-Myc antibody or normal rabbit IgG. Immunoprecipitated chromatin was quantified with RT-PCR. (C) NRVMs were pretreated with losartan (10 µmol/L, lane 3), NAC (10 mmol/L, lane 4), apocynin (100 µmol/L, lane 5), rotenone (10 µmol/L, lane 6), gp91 ds-tat (10 µmol/L, lane 7) or PD98059 (10 µmol/L, lane 8) for 1 h, and then stimulated with 0.1 µmol/L AngII for 48 h. DNA binding activity of c-Myc was determined by EMSA with nuclear extracts of NRVMs and DNA probes. Lane 1, control; lane 2, AngII; lane 3–8, inhibitors; lane 9, with 100-fold molar excess of unlabeled oligonucleotide; lane 10, with 100-fold molar excess of unlabeled mutant oligonucleotide.

Figure 6
Figure 6. AngII decreases AdipoR1 expression in a c-Myc-dependent manner.

(A) NRVMs were transfected with c-Myc or control siRNA (100 nmol/L) for 48 h. Western blot was performed to detect levels of c-Myc and actin. (B) NRVMs were transfected with c-Myc or control siRNA (100 nmol/L) for 48 h and serum-deprived for 24 h, followed by incubation with 0.1 µmol/L AngII for 48 h. The level of AdipoR1 protein was analyzed by Western blot analysis with actin as an internal control. Data represent mean ± SE of three independent experiments. *P<0.05, **P<0.01 vs. control. ## P<0.01 vs. AngII.

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This work was supported by grants from the National Nature Science Foundation of China (Nos. 30871014 and 30973316) and the National Basic Research Program of China (973 Program 2007CB512004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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