Activation of AMP-Activated Protein Kinase Enhances Angiotensin II–Induced Proliferation in Cardiac Fibroblasts
AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic regulator of cellular enzymes involved in carbohydrate and fat metabolism, which regulate ATP conservation and synthesis. Here, we investigated whether AMPK signaling has a role in the regulation of angiotensin II (Ang II)–induced proliferation in rat cardiac fibroblasts. Aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR) activated AMPK in rat cardiac fibroblasts and increased Ang II–induced extracellular signal–regulated kinase 1/2 phosphorylation and activity. AICAR also increased Ang II–induced c-fos mRNA expression in the cells. [3H]-thymidine and [3H]-proline incorporation by cardiac fibroblasts treated with Ang II was enhanced when the cells were pretreated with AICAR. Inhibition of AMPK by small interfering RNA for AMPKα1 suppressed Ang II–induced extracellular signal–regulated kinase activity, c-fos mRNA expression, and cell proliferation. Treatment of rats with AICAR (1 mg/g body weight per day) for 1 week significantly enhanced Ang II–induced hypertrophy of the myocardium. Our findings indicate that AMPK works as a stimulator of the Ang II–induced proliferative pathway in cardiac fibroblasts. Inhibition of AMPK signaling might serve as a new therapeutic target of remodeling of the hypertrophic myocardium.
AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic regulator of cellular enzymes involved in carbohydrate and fat metabolism, which regulate ATP conservation and synthesis.1 AMPK is activated by conditions that increase the AMP:ATP ratio, such as exercise and metabolic stress. AMPK has been most extensively examined under conditions of stress, such as exercise and hypoxia–ischemia. When the AMP:ATP ratio increases, AMPK is activated by AMPK kinase to combine with AMP, thus inducing a conformational change and decreasing the AMP:ATP ratio by switching off ATP-consuming pathways and switching on ATP-generating pathways.1
Considerable information exists regarding the regulation of AMPK activation in a variety of cell types, including cardiac cells.2,3 There is a correlation between AMPK activation and the development of cardiac hypertrophy.4,5 Pressure overload–induced hypertrophic cardiomyopathy is associated with increased AMPK activity.4 In addition, AMPK plays a role in familial Wolff—Parkinson–White syndrome and hypertrophic cardiomyopathy via mutations in PRKAG2, a gene encoding the γ2 subunit of AMPK.6–8 On the other hand, recent evidence indicates that AMPK activation inhibits proteins synthesis9,10 and might, thus, be expected to reduce the development of hypertrophy. Although much has been learned about the role of AMPK in the development of cardiac hypertrophy, the molecular mechanism by which AMPK regulates cardiac hypertrophy is still unclear.
Interstitial fibroblast proliferation and collagen accumulation is associated with compensatory remodeling of the hypertrophic myocardium,11,12 and structural remodeling leads to diastolic and systolic dysfunction.13 Angiotensin (Ang) II is closely involved in cardiac remodeling by stimulating hyperplastic growth of cardiac fibroblasts14 and the synthesis of extracellular matrix proteins.12 Therefore, we investigated the role of AMPK in cardiac remodeling using cardiac fibroblasts. We used 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR), which is an adenosine analog, as well as cell-permeable activator of AMPK and a small interfering RNA (siRNA) for AMPK, which is an AMPK inhibitor. We examined the effects of both the AMPK activator and inhibitor on the extracellular signal–regulated kinase (ERK) 1/2 pathway and DNA and collagen synthesis in Ang II–stimulated cultured rat cardiac fibroblasts. We additionally examined the effect of AICAR on the development of myocardial hypertrophy in Ang II–infused rats.
Cardiac fibroblasts were prepared from the ventricles of 1- to 2-day-old Wistar rats and grown as described previously.15,16 Subcultured fibroblasts from passages 4 to 6 were used in this experiment, and they were >99% positive for immunostaining with vimentin antibody. In addition, they were negative for desmin (for myocytes), smooth muscle actin (for vascular smooth muscle cells), and a polyclonal antibody against von Willebrand factor (for endothelial cells). Subconfluent cells were serum starved for 48 hours and used for the experiments.
One day before transfection, plates were inoculated with an appropriate number of cardiac fibroblasts in serum-containing medium to ensure 50% to 70% confluence the following day. AMPKα1 siRNA (Santa Cruz Biotechnology, Inc) mixed with siLentFect (Bio-Rad Laboratories) was added to cells to make a final concentration of 10 nM/L. Twenty-four hours after transfection, the cells were serum starved for another 24 hours. A ≈10% to 20% decrease in Ang II–activated phospho-AMPK was observed in transfected cells compared with control cells.
Analysis of DNA and Collagen Synthesis
The effects of AICAR (Toronto Research Chemicals Inc) on DNA and collagen synthesis in cardiac fibroblasts were evaluated by incorporation of [3H]-thymidine and [3H]-proline into cells, respectively, as described previously.15,16 To examine collagen synthesis, 0.5 μCi of [3H]-proline was added after treatment with Ang II (Sigma Chemical Co), and the cells were incubated for 24 hours. To examine DNA synthesis, 0.5 μCi of [3H]-thymidine was added 12 hours after treatment with Ang II, and the cells were incubated for 12 hours. After labeling, the cells were rinsed twice with cold PBS and incubated with 10% trichloroacetic acid at 4°C for 30 minutes. The precipitates were then washed twice with cold 95% ethanol and solubilized in 1 mol/L NaOH. The radioactivity of aliquots of trichloroacetic acid–insoluble material was determined using a liquid scintillation counter.
Cardiac fibroblasts treated with Ang II in the presence or absence of AICAR were lysed using cell lysis buffer (Cell Signaling) with 1 mmol/L PMSF. The protein concentration within each sample was measured using a Bio-Rad detergent-compatible protein assay. Subsequently, β-mercaptoethanol was added at a final concentration of 1%, after which each sample was denatured by boiling for 3 minutes. Samples containing 15 μg of protein were resolved by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad) incubated with Phospho-AMPK antibody, AMPK antibody, Phospho-ERK antibody, and ERK antibody (1:1000, Cell Signaling). The binding of each of these antibodies was detected using sheep anti-rabbit IgG horseradish peroxidase (1:5000) and the ECL Plus system (Amersham).
Cardiac fibroblasts grown on 24-well plates were stimulated with agonists at 37°C in serum-free DMEM for 48 hours. Each reaction was terminated by replacement of the medium with ice-cold lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 20 mmol/L NaCl, 2 mmol/L EGTA, 2 mmol/L dithiothreitol, 1 mmol/L Na3VO4, 1 mmol/L PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin]. After a brief period of sonication, each sample was centrifuged for 5 minutes at 14 000g, after which the supernatant was assayed for ERK activity with an assay kit (Amersham) measuring incorporation of [γ-33P]ATP into a synthetic peptide (KRELVEPLTPAGEAPNQALLR) as a specific ERK substrate. Each reaction mixture contained the cell lysate in 75 mmol/L HEPES (pH 7.4), containing 1.2 mmol/L MgCl2, 2 mmol/L substrate peptide, and 1.2 mmol/L ATP, along with 1 μCi of [γ-33P]ATP, and each reaction took place for 30 minutes at 30°C, as described previously.17 The resultant solution was applied to a phosphocellulose membrane and extensively washed in 1% acetic acid and then in H2O. Radioactivity was measured by liquid scintillation counting.
For quantitative measurement of mRNA, 2 μg of total RNA was treated with DNase I for 15 minutes and subsequently used for cDNA synthesis. The PCR reactions with the cDNA were carried out in LineGene system (BioFlux) under the following conditions: 95°C for 5 minutes, 40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s.
The experiments were reviewed and approved by the Committee on Ethics of Animal Experiments and were conducted in accordance with the Guidelines for Animal Experiments, Dokkyo University Faculty of Medicine.
Seven-week-old male Sprague-Dawley rats (Tokyo Experimental Animals, Tokyo, Japan) were randomly assigned to 1 of 3 experimental groups of 6 rats. Vehicle (distilled water) was infused into the control group. Ang II was infused without AICAR (Ang II groups) or with AICAR (Ang II/AICAR group). Ang II was infused subcutaneously using an osmotic pump (model 2002, Alza Corporation) at a dose of 300 ng/kg per minute for 7 days. Rats were dosed by oral lavage daily for 7 days with vehicle (Ang II group; 0.5% methylcellulose with 0.1% Tween 80) or AICAR (Ang II/AICAR group; 1 mg/g body weight per day) in vehicle starting 24 hours before Ang II infusion. Rats were anesthetized by intraperitoneal injection of pentobarbital 24 hours after the last AICAR treatment. The hearts were obtained and weighed before histochemical analysis.
The results are expressed as mean±SE. ANOVA and Fisher’s least-significant difference test were used for multigroup comparisons, with P<0.05 considered significant.
AMPK Activated by AICAR Increases Ang II–Induced ERK Pathway Activation
AICAR induced AMPK phosphrylation after incubation for 1 hour and remained phosphorylated for ≥2 hours (Figure 1A). Transfection of siRNA for AMPK into rat cardiac fibroblasts markedly inhibited AMPK expression in the absence and presence of AICAR (Figure 1A).
Ang II-induced ERK phosphorylation reached a maximum after 15 minutes (Figure 1B). We examined the effect of AICAR on Ang II–induced ERK phosphorylation. When cells were preincubated with AICAR for 1 hour, Ang II–induced ERK phosphorylation was clearly enhanced (Figure 1B).
Next, we examined the effect of overexpression of siRNA for AMPK on Ang II–induced ERK phosphorylation. Transfection of siRNA into rat cardiac fibroblasts markedly inhibited ERK phosphorylation both in the absence and presence of AICAR after Ang II stimulation (Figure 1C).
We additionally examined Ang II–induced ERK activity in cardiac fibroblasts. ERK activity was substantially induced by Ang II, which was significantly enhanced by AICAR treatment (Figure 1D). On the other hand, siRNA treatment markedly decreased Ang II–induced ERK activity (Figure 1D).
AMPK Activated by AICAR Enhances Ang II–Induced c-fos mRNA Expression
We examined the effect of AICAR on Ang II–induced c-fos mRNA expression. Very low levels of c-fos mRNA were observed in untreated cells, but Ang II treatment for 1 hour clearly increased the mRNA levels (Figure 1). When cells were stimulated with Ang II after preincubation with AICAR, the induction of c-fos mRNA was substantially enhanced. The enhanced mRNA levels were completely prevented by olmesartan (AT1 antagonist) but not by PD123319 (AT2 antagonist). The MAPK kinase (MEK) inhibitor PD98059 markedly inhibited the Ang II–induced c-fos mRNA expression. However, in cells pretreated with AICAR, the inhibitory effect of PD98059 on the Ang II–induced c-fos mRNA expression was clearly blunted (Figure 2).
AMPK Enhances Ang II–Induced [3H]-Thymidine and [3H]-Proline Incorporation in Cardiac Fibroblasts
We observed that AMPK increased the activity of the MAPK/ERK pathway, thus, we next investigated whether AMPK activation might enhance Ang II–induced cell proliferation and collagen synthesis in cardiac fibroblasts. We measured incorporation of [3H]-thymidine and [3H]-proline 24 hours after administration of 10−6 mol/L Ang II. In control cells, Ang II stimulation increased [3H]-thymidine and [3H]-proline incorporation by 1.6 and 1.2 times, respectively. When AMPK was activated by pretreatment with AICAR, dose-dependent enhancement of [3H]-thymidine and [3H]-proline incorporation was observed (Figure 3). We also examined the effect of suppression of AMPK activity by transfection of siRNA on Ang II–induced [3H]-thymidine and [3H]-proline incorporation, which were suppressed by 50% and 30%, respectively, compared with controls (Figure 3). Olmesartan, but not PD123319, completely inhibited Ang II–induced [3H]-thymidine and [3H]-proline incorporation (data not shown). These results are compatible with the consensus that AT1R is the main receptor subtype responsible for transducing the Ang II signal for cell proliferation.
In Vivo Administration of AICAR Exacerbates Myocardial Hypertrophy
Finally, we examined the in vivo effect of AICAR on cardiac weight in Ang II–infused rats, followed by histochemical analysis. At the same time, we examined whether administration of AICAR might activate AMPK within the rat heart by immunoblot analysis.
The mean heart weight index (mg/kg body weight) was significantly greater in the Ang II group (3.64±0.11) compared with the control group (3.16±0.13). The development of myocardial hypertrophy was enhanced by concomitant treatment with AICAR (Ang II/AICAR group; 3.88±0.14), which was significantly greater than the Ang II group. A representative section of the heart is shown in Figure 4, indicating that Ang II–induced myocardial hypertrophy was enhanced by cotreatment with AICAR. AICAR significantly increased AMPK activity in the heart, suggesting a specific effect of AICAR on AMPK activation.
AMPK is a stress-activated protein kinase that functions as a metabolic sensor of cellular ATP levels.1 In the present study, we demonstrated that AICAR activates AMPK in rat cardiac fibroblasts and enhances Ang II–induced ERK phosphorylation and activation. To verify the specificity for AMPK activation, we transfected siRNA for AMPK to confirm inhibition of Ang II–induced ERK phosphorylation and activation.
AICAR pretreatment significantly augmented Ang II–induced [3H]-thymidine and [3H]-proline incorporation. In addition, inhibition of AMPK by siRNA inhibited Ang II–induced [3H]-thymidine and [3H]-proline incorporation. These results suggest that AMPK enhances cell proliferation and collagen synthesis induced by Ang II. Furthermore, because enhanced Ang II–induced c-fos mRNA levels were observed in cardiac fibroblasts after pretreatment with AICAR, which was inhibited by AT1R antagonist but not by MEK inhibitor, the AMPK target likely falls between the AT1R and MEK, leading to the activation of ERK → Fos → cell proliferation.
In our animal study, we observed an increased mean heart weight index (mg/kg body weight) among Ang II–treated rats treated with AICAR compared with rats treated with Ang II alone. Ang II resulted in cardiomegaly, which was enhanced by concurrent treatment with AICAR. Significantly increased blood pressure levels were observed in Ang II–treated rats; however, they did not differ significantly from rats cotreated with AICAR. We demonstrated synchronization of Ang II–induced AMPK activation with ERK signaling and showed that AMPK augments Ang II–induced cell proliferation. Thus, the effect of AICAR on cardiac hypertrophy in vivo was not associated with blood pressure but appeared to result from superactivation of the ERK-proliferative pathway shown in cell culture experiments.
Two thirds of the myocardial cell population is composed of nonmyocyte cells, the majority of which are fibroblasts, in the situation of long-standing renovascular hypertension with marked remodeling of the heart.12 Cardiac fibroblasts are responsible for the production and deposition of extracellular matrix proteins, such as fibronectin and collagen types 1 and 3, which was reported to be induced in response to Ang II.12,18 It has been reported that Ang II receptors are localized predominantly on cardiac fibroblasts and that Ang II can indirectly stimulate hypertrophy of cardiac myocytes by stimulating cardiac fibroblasts to produce transferable factors.19 Therefore, the increase by AICAR in incorporation of thymidine and proline into cardiac fibroblasts may lead to an additional myocardial hypertrophy. We did additional experiments using human cardiac fibroblasts and rat cardiac myocytes to see whether AICAR increases c-Fos mRNA induction by Ang II. Unstimulated human cardiac fibroblasts express very low levels of c-Fos mRNA, but it was induced by Ang II and was additionally enhanced by AICAR. On the other hand, cardiac myocytes, which are beating, express low levels of c-Fos mRNA, and Ang II did not change the levels of the c-Fos mRNA. However, the levels of c-Fos mRNA substantially increased by coaddition of AICAR. Thus, the study of 2 different cell types also suggests that AICAR makes myocardial cells into myocardial hypertrophy. Recently, it has been discovered that AMPK inhibits Ang II–stimulated vascular smooth muscle cell proliferation.20 Several reports indicate that the antidiabetic adipocytokine adiponectin, which activates AMPK, has antiatherosclerotic effects.21–23 Moreover, adiponectin activates AMPK and inhibits agonist-stimulated myocardial hypertrophy and ERK activation, as well as ERK transduction. A dominant-negative form of AMPK reverses this effect in cardiac myocytes, suggesting that adiponectin inhibits hypertrophic signaling in the myocardium through activation of AMPK signaling.24 On the other hand, pressure overload–induced hypertrophy is associated with increased AMPK activity.2 In addition, AMPK plays a role in familial Wolff–Parkinson–White syndrome and hypertrophic cardiomyopathy via mutations in PRKAG2, a gene encoding the γ2 subunit of AMPK.4–6 Although the mechanism by which different cell types respond to AMPK activation remains to be elucidated, all of the data combined suggest that AMPK might function to regulate cell proliferation, including cardiac fibroblasts, and that inhibition of AMPK signaling might serve as a new therapeutic target of cardiac remodeling in patients with hypertrophic cardiomyopathy.
Our data suggest that AMPK signaling has a role in the regulation of Ang II–induced proliferation in cardiac fibroblasts. However, the in vivo model may not be desirable, because fibroblast proliferation might not be a major early event in myocardial hypertrophy in this short-term model. Growth of cardiomyocytes, as well as fibroblast proliferation, may explain the in vivo relevance of the findings. Indeed, the levels of c-Fos mRNA in cardiac myocytes substantially increased by the coaddition of AICAR with Ang II. These results contribute to our understanding of the mechanisms involved in myocardial hypertrophy and may provide a basis for possible strategies to prevent or reverse cardiac remodeling.
We are grateful to Hiroko Satoh for technical assistance. We also thank Noriko Suzuki and Fumie Yokotsuka for histological investigation.
- Received September 29, 2005.
- Revision received October 17, 2005.
- Accepted November 18, 2005.
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