The authors have declared that no competing interests exist.
Conceived and designed the experiments: LLW LL ZGZ. Performed the experiments: LL ZGZ HL CW FYF JYW. Analyzed the data: LL ZGZ. Contributed reagents/materials/analysis tools: LPW WGZ. Wrote the paper: LL LLW ZGZ.
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
Adiponectin is an abundant adipose tissue-derived protein with important metabolic modulation and energy homeostasis effects
Two types of adiponectin receptors (AdipoRs), AdipoR1 and AdipoR2, mediate most effects of adiponectin via activating adenosine monophosphate-activated protein kinase (AMPK)
Angiotensin II (AngII), the major component of renin-angiotensin system (RAS), exerts vasoconstrictive, growth-promoting, and remodeling effects on the cardiovascular system
The present study was designed to investigate the effect of AngII on AdipoRs expression in rats exposed to continuous infusion of AngII and in cultured neonatal rat cardiomyocytes. We also explored the possible molecular mechanism by which AngII regulates AdipoRs expression.
AngII, PD123319, CGP42112A, N-acetyl cysteine (NAC), apocynin, retenone, allopurinol, PD98059, SB202190, and SP600125 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Losartan was from Merck & Co. (Whitehouse Station, NJ, USA). gp91 ds-tat and scrambled gp91 ds-tat were from Anaspec (San Jose, CA, USA). Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for phospho- and total AMPK, and phospho- and total acetyl coenzyme A carboxylase (ACC) were from Cell Signaling Technology (Beverly, MA, USA). Anti-c-Myc antibody was from Upstate (Billerica, MA, USA). Anti-signal transducer and activator of transcription (STAT) 5 antibody was from Abcam (Cambridge, UK).
All experimental procedures were approved by the Ethics Committee of Animal Research, Peking University Health Science Center, and the investigation conformed to the guidelines of the National Institutes of Health for the care and use of laboratory animals. Male Sprague-Dawley (SD) rats weighing 250 to 280 g were randomly divided into control, AngII, and losartan groups. An osmotic mini-pump (model 2004, Durect Corp., Cupertino, CA, USA) was subcutaneously embedded in rats under anesthesia with sodium pentobarbital (50 mg/kg, IP). AngII (400 ng/kg/min) or normal saline was infused constantly for 28 days. In the losartan group, rats also received losartan (10 mg/kg/day orally) during AngII infusion. At day 28, rats were anesthetized by sodium pentobarbital (50 mg/kg, IP). The right carotid artery was cannulated with polyethylene tubing connected to a Model TCB-500 transducer control unit (Millar Instruments, Houston, TX, USA). Hemodynamic parameters were recorded on a PowerLab data-acquisition system (ADInstruments, Sydney, Australia). Blood was collected and plasma was separated by centrifugation. Hearts, blood vessels, skeletal muscle, and adipose tissue were then excised for further investigation.
Plasma total adiponectin was determined with a rat adiponectin ELISA kit (Phoenix Pharmaceuticals Inc., Belmont, CA, USA).
Primary neonatal rat ventricular myocytes (NRVMs) was cultured as described previously
Total RNA of myocardial tissues or NRVMs was isolated by use of Trizol reagent (Invitrogen) and cDNA was generated from total RNA by use of the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada). qRT-PCR was performed using following primer sets: AdipoR1 (forward,
Myocardial tissues or NRVMs were lysed in a buffer containing 50 mM Tris-HCl, pH 7.2, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 40 mM NaF, 2.175 mM sodium orthovanadate, 0.1% SDS, 0.1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged at 10 000×g for 10 min (4°C) and the supernatant was collected. Equal amounts of protein (50 µg), assayed using the Lowry's method
NRVMs were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 in PBS. NRVMs were stained with anti-AdipoR1 or AdipoR2 antibody overnight at 4°C, then fluorescence-labeled secondary antibody for 2 h at 37°C. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Fluorescence images were captured on the Olympus confocal microscope (Olympus Corp., Tokyo, Japan). AdipoRs-positive areas were quantified by use of Image J software (National Institute of Health, Bethesda, MD, USA). Fluorescence density of AdipoRs was expressed as the positive area corrected for the number of nuclei.
Ventricles were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Deparaffined sections (5 µm thickness) were incubated with primary antibody against AdipoR1 or AdipoR2 overnight at 4°C, then horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactions were visualized with 3-3′ diaminobenzidine tetrahydrochloride. The nuclei were counterstained with hematoxylin. Negative controls involved omission of primary antibodies. Microscopy images were analyzed by use of Leica QWin image analysis software (Leica, Wetzlar, Germany). Staining density was expressed as positive area corrected for the numbers of nuclei.
Intracellular ROS level was determined by using the cell-permeable, redox-sensitive fluorophore, 2′,7′-dichlorofluorescin diacetate (DCF-DA) (Molecular Probes, Eugene, OR, USA). NRVMs were incubated with 20 µM DCF-DA for 30 min and DCF fluorescence was visualized using the Olympus confocal microscope and analyzed with Image J software. All experiments were done with minimal exposure to light, and fluorescence was normalized to cell count.
ChIP assay was performed as described by Wu
Nuclear protein fractions were separated as described
NRVMs were seeded at a density of 3×105 cells per well of six-well plates. After 48–72 h culture, cells were transfected with siRNA of interest by use of Lipofectamine 2000 (Invitrogen). The potent siRNA for rat c-Myc was designed by use of the siRNA Target Finder (Ambion Inc., Austin, TX, USA), based on the Rattus norvegicus mRNA sequences deposited at the NIH-PubMed database, and were submitted to BLAST analysis to assure specificity. The potent rat c-Myc siRNA
Deparaffined heart sections (5 µm thickness) were stained with hematoxylin and eosin for routine histological examination. Images were captured from six hearts in each group and cardiac myocyte cross-sectional surface area was evaluated by use of Leica QWin image analysis software. One hundred myocytes per heart were counted, and the average area was determined.
Data are expressed as mean ± SE. Data were compared by one-way ANOVA for multiple groups, followed by Bonferroni's tests by use of GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).
Hemodynamic parameters on day 28 were shown in
Plasma adiponectin levels in AngII-infused rats were significantly lower than that in controls, and losartan treatment prevented the AngII-induced decrease in circulating adiponectin levels (
(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. **
There was no significant difference of AdipoRs protein expression in skeletal muscle and adipose tissue among the control, AngII, and losartan groups (
To determine the effect of AngII on AdipoRs expression
(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. **
To determine the receptor subtypes mediating the inhibitory effect of AngII on AdipoR1 expression, NRVMs were pretreated with 10 µmol/L losartan or 1 µmol/L PD123319, an AT2 receptor antagonist, for 1 h and then stimulated with 0.1 µmol/L AngII for 48 h. Expression of AdipoR1 mRNA and protein was downregulated by AngII, which was reversed significantly when pretreated with losartan. However, the suppressive effect of AngII on AdipoR1 expression was not affected by PD123319. Furthermore, CGP42112A, a specific AT2 receptor agonist, did not change the expression of AdipoR1 (
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. *
To explore the role of mitogen-activated protein kinase (MAPK) in AngII-reduced AdipoR1 expression, we employed SB202190 (5 µmol/L), a selective p38MAPK inhibitor, PD98059 (10 µmol/L), an ERK1/2 upstream kinase inhibitor, and SP600125 (10 µmol/L), a c-Jun-N-terminal kinase (JNK) inhibitor to preincubate NRVMs for 1 h, and then stimulated with 0.1 µmol/L AngII for 48 h. PD98059 reversed AngII-reduced AdipoR1 expression. SB202190 and SP600125 did not affect AngII-induced AdipoR1 downregulation (
(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. *
We explored whether ROS was involved in the activation of ERK1/2 and downregulation of AdipoR1 in response to AngII. Fluorescence density of DCF was significantly increased after AngII treatment for 30 min. AngII-increased ROS production was eliminated by the ROS scavenger NAC (10 mmol/L), the NADPH oxidase inhibitor apocynin (100 µmol/L), and the inhibitor of the mitochondrial electron transport chain complex I rotenone (10 µmol/L), but not by the inhibitor of xanthine oxidase allopurinol (100 µmol/L) (
The catalytic subunit gp91phox (Nox2), a major isoform of NADPH oxidase expressed in cardiomyocytes, is critical for cardiac hypertrophy in response to chronic AngII infusion
To further reveal the transcription mechanism of AngII-mediated AdipoR1 expression, we screened the DNA sequence of rat AdipoR1 promoter region by use of Genomatix from the UCSC Genome Browser Database (
(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.
Sequence | Chromosomal Location | |
c-Myc site 1 |
|
−1051 to −1039 |
c-Myc site 2 |
|
−2036 to −2024 |
c-Myc site 3 |
|
−2515 to −2503 |
STAT5 site 1 |
|
−2636 to −2618 |
STAT5 site 2 |
|
−2830 to −2812 |
NF-κB site |
|
−225 to −213 |
To further examine the effect of c-Myc on AngII-reduced AdipoR1 expression, NRVMs were transfected with control or c-Myc siRNA (100 nmol/L) for 48 h and serum-deprived for 24 h, then incubated with 0.1 µmol/L AngII for 48 h. c-Myc siRNA greatly reduced c-Myc protein level (
(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. *
In the present study, we demonstrated that AngII reduced AdipoR1 but not AdipoR2 expression both in hearts of AngII-infused rats and in cultured neonatal rat cardiomyocytes. We also found that AT1 receptor, ROS derived from NADPH oxidase and mitochondria, and ERK1/2 were critical for AngII-reduced AdipoR1 expression. The recruitment of c-Myc to AdipoR1 promoter region was required for the transcription modulation of AngII on AdipoR1 expression. Moreover, the AT1 receptor/ROS/ERK1/2 pathway was involved in AngII-induced DNA binding activity of c-Myc. These results suggest that the downregulation of cardiac AdipoR1 by AngII may play an important role in the progression of cardiac remodeling. This research focused on the mechanism involved in AngII-reduced AdipoR1 expression in cardiomyocytes will hopefully lead to novel strategies for clinical cardioprotection.
Adiponectin is an endogenous protective protein that acts as a modulator in energy metabolism and cardiovascular function
AngII binds to and activates AT1 and AT2 receptors, which seem to induce phenotypically opposite responses
AngII regulates cell behavior through several mechanisms downstream of AT1 receptor, including stimulation of MAPK cascades. ERK1/2, p38MAPK, and JNK are three major members of the MAPK family, but the exact signal molecule responsible for AdipoR1 expression in this process is still poorly understood. We found that the suppressive effect of AngII on AdipoR1 expression was prevented by PD98059, but not by SB202190 and SP600125, which indicates that ERK1/2 is critical for AngII-reduced AdipoR1 expression in cardiomyocytes.
Increased ROS production mediates the hypertrophic response in cardiomyocytes to stretch or to neurohormonal stimuli such as AngII
Although AdipoRs expression is mediated by various physiological and pathological factors, the precise mechanism involved in AdipoRs transcription regulation has not been fully evaluated. Recently, an insulin-responsive repressor element, termed nuclear inhibitory protein, has been identified in the AdipoR1 promoter region of C2C12 cells
In summary, we demonstrate that AngII reduces the expression of AdipoR1 but not AdipoR2 in hearts of AngII-infused rat model and in cultured neonatal cardiomyocytes. AngII decreases AdipoR1 expression through its AT1 receptor. ROS generated from NADPH oxidase, especially Nox2, and mitochondria and the sequential activation of ERK1/2 are involved in AngII-reduced AdipoR1 expression. Moreover, c-Myc plays an important role in AngII-mediated AdipoR1 transcription regulation in cardiomyocytes through binding to AdipoR1 promoter directly. The AT1 receptor/ROS/ERK1/2 pathway is required for AngII-induced DNA binding activity of c-Myc. These findings may improve our understanding of the molecular mechanisms involved in cardiac hypertrophy and provide new insights into future therapeutic targets for cardiac remodeling.
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