行政院國家科學委員會專題研究計畫 成果報告
C 急性反應蛋白在 HL1 細胞株及新生心房纖維母細胞向上調 控血管收縮素 II 第一型接受體(第 2 年)
研究成果報告(完整版)
計 畫 類 別 ： 個別型
計 畫 編 號 ： NSC 99-2314-B-040-020-MY2
執 行 期 間 ： 100 年 08 月 01 日至 101 年 07 月 31 日 執 行 單 位 ： 中山醫學大學醫學系
計 畫 主 持 人 ： 翁國昌
計畫參與人員： 碩士級-專任助理人員：朱小瓀
公 開 資 訊 ： 本計畫涉及專利或其他智慧財產權，2 年後可公開查詢
中 華 民 國 101 年 10 月 29 日
中 文 摘 要 ： 心房顫動是臨床最常見的心律不整。在一般人群的總患病率 在 0.4％，隨著年齡增加，心房顫動有逐漸增加的趨勢，在 75 歲以上人群可達 10％。心房顫動會明顯增加腦中風及心臟 衰竭甚至死亡的機會，因此心房顫動是消耗社會成本很重的 一種心律不整的疾病。近年來有許多研究指出腎素-血管收縮 素-留鹽激素系統 (renin-angiotensin-aldosterone
system, RAAS)與發炎反應及 AF 有潛在的關連性。最近的研 究發現，在老鼠體內，心房壓力上升時，會促使心房血管收 縮素上升，進而造成心房結構重構，使心房纖維化(AF)及心 房擴大。心房纖維顫動的發生和腎素-血管張力素系統有很大 的關係，血管張力素可使得心房纖維顫動更容易發生及維 持。當血管收縮素結合在血管收縮素 II 第一型接受體時，會 促使血管收縮使血壓上升，並促進腎上腺增加留鹽激素的產 生和分泌，留鹽激素亦會調控血管收縮素 II 第一型接受體的 表現。本研究的目的，主要在於留鹽激素和心房纖維顫動發 生機轉的關係。探討 Aldosterone 是否造成心肌細胞內 AT1； mineralocorticoid receptors (MR)及纖維化指標分 子等蛋白表現量的改變，進而影響心臟之功能。在本研究報 告，我們利用細胞模式，來探討留鹽激素在促進心房纖維顫 動之可能機制。研究結果顯示，在培養 HL-1 心房細胞時，加 入留鹽激素可以促使 MR、AT1 及纖維化指標分子蛋白表現增 加，並且觀察 MAPK 活化的表現；在加入留鹽激素拮抗劑 eplerenone 或血管收縮素第一型受體阻斷劑 losartan 會抑 制留鹽激素促使纖維化指標分子（COL1A, COL3A, TGF-β1, α-SMA）蛋白表現增加，並觀測到其表現是經由 MAPK 路徑調 控；利用 MAPK 磷酸化抑制劑處理，發現阻斷留鹽激素路徑的 活化。因此留鹽激素在心房纖維顫動病變可能扮演重要的角 色，其機轉和 MAPK 之相關訊息傳導路徑有關。
中文關鍵詞： 心房顫動，留鹽激素，血管收縮素 II 第一型受體，HL-1 心 房細胞
英 文 摘 要 ： Objectives To investigate the regulation of
aldosterone- mineralocorticoid receptor (MR) on the expression of fibrotic marker proteins in cultured HL-1 cardiomyocytes.
Background Aldosterone is increasingly recognized for its involvement in atrial structural remodeling.
However, the precise molecular mechanisms and signal pathways underlying aldosterone-induced atrial
fibrosis are unknown.
Methods Western blotting was used to investigate the effects of aldosterone on the expression of MR, angiotensin II type I receptor (AT1), mitogen- activated protein kinases (MAPKs), and fibrotic marker proteins in cultured HL-1 cardiomyocytes.
Results Aldosterone concentration and time-
dependently upregulated MR and AT1 expression； and time-dependently increased activation of the
extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38MAPK pathways, and the protein expression of collagen 1A and 3A (COL1A and COL3A), transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA). Pre-treatment with eplerenone (10−0 M) prevented the increased expression of MR, MAPK signals and the above
profibrotic molecules, but amplified the increase in AT1 level stimulated by aldosterone (10− M). Pre- treatment with losartan (10−0 M) or MAPK pathway inhibitors (U0126 or SP600125) abolished aldosterone- induced MR upregulation and significantly inhibited the expression of the above fibrotic marker proteins, indicating the critical role of MR and the
requirement for active AT1 in the development of aldosterone-induced atrial fibrosis.
Conclusions Elevated MR activity plays a central role in aldosterone-mediated activation of the MAPK
signaling pathway and subsequent profibrotic effects in HL-1 atrial cells. MR/AT1 and the MAPK signaling pathway interact to trigger the molecular mechanism underlying the aldosterone-induced atrial fibrotic response.
英文關鍵詞： aldosterone, angiotensin, fibrosis, atrial fibrillation, signal transduction
Cross-Talk Between Mineralocorticoid Receptor/Angiotensin II Type 1 Receptor and Mitogen-Activated Protein Kinase Pathways Underlies Aldosterone-Induced Atrial Fibrotic Responses in HL-1 Cardiomyocytes
Chin-Feng Tsai,1 MD, PhD;Shun-Fa Yang,2,3 PhD; Hsiao-Ju Chu,3 MS; Kwo-Chang Ueng,1 MD, PhD
1Division of Cardiology, Department of Internal Medicine, Chung Shan Medical University Hospital; School of Medicine, Chung Shan Medical University, Taichung, Taiwan;
2Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
3Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
The first two authors contributed equally.
Running title: Aldosterone-Induced Atrial Profibrosis
This work was supported by the grants from the National Science Council (NSC) (NSC-99-2314-B-040-020-MY2, NSC-101-2314-B-040-018-MY3), Taiwan, R.O.C.
Kwo-Chang Ueng, MD, PhD Address for correspondence:
Department of Internal Medicine, Chung Shan Medical University Hospital School of Medicine, Chung Shan Medical University
No. 110, Sec. 1, Jianguo N. Road, Taichung City 40201, Taiwan Tel: 886-4-24739595 ext. 34523, Fax: 886-4-24739220
Email: [email protected]
Structured Abstract
Objectives To investigate the regulation of aldosterone- mineralocorticoid receptor (MR) on the expression of fibrotic marker proteins in cultured HL-1 cardiomyocytes.
Background Aldosterone is increasingly recognized for its involvement in atrial structural remodeling. However, the precise molecular mechanisms and signal pathways underlying aldosterone-induced atrial fibrosis are unknown.
Methods Western blotting was used to investigate the effects of aldosterone on the expression of MR, angiotensin II type I receptor (AT1), mitogen-activated protein kinases (MAPKs), and fibrotic marker proteins in cultured HL-1 cardiomyocytes.
Results Aldosterone concentration and time-dependently upregulated MR and AT1 expression;
and time-dependently increased activation of the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38MAPK pathways, and the protein expression of collagen 1A and 3A (COL1A and COL3A), transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA). Pre-treatment with eplerenone (10−10 M) prevented the increased
expression of MR, MAPK signals and the above profibrotic molecules, but amplified the increase in AT1 level stimulated by aldosterone (10−6 M). Pre-treatment with losartan (10−10 M) or MAPK pathway inhibitors (U0126 or SP600125) abolished aldosterone-induced MR
upregulation and significantly inhibited the expression of the above fibrotic marker proteins, indicating the critical role of MR and the requirement for active AT1 in the development of aldosterone-induced atrial fibrosis.
Conclusions Elevated MR activity plays a central role in aldosterone-mediated activation of the MAPK signaling pathway and subsequent profibrotic effects in HL-1 atrial cells. MR/AT1 and the MAPK signaling pathway interact to trigger the molecular mechanism underlying the
aldosterone-induced atrial fibrotic response.
Key Words: aldosterone, angiotensin, fibrosis, atrial fibrillation, signal transduction
Abbreviations Lists
AF, atrial fibrillation; AT1, angiotensin II type 1 receptor; COL,collagen; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MR, mineralocorticoid receptor; SMA, smooth muscle actin; TGF, transforming growth factor
Introduction
Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical medicine. AF is a progressive disease. Extensive evidence indicates that structural remodeling, particularly atrial fibrosis, is an important contributor to the AF substrate (1, 2).In animal models,
experimentally-induced atrial fibrosis increased the likelihood of AF (3, 4). Atrial tissue samples from patients with isolated AF also showed increased fibrosis (5). Interstitial fibrosis is promoted by the increased expression of profibrotic cytokines (e.g., transforming growth factor [TGF]-β and α-smooth muscle actin [SMA]), and more pronounced simultaneous expression of collagen I and III (COL1A and COL3A) (2). It has been hypothesized that atrial fibrosis may facilitate local intra-atrial conduction block and atrial heterogeneity, thereby enhancing the propensity to AF occurrence and self-perpetuation (2, 6). However, the regulatory mechanisms and signaling pathways involved in the development of atrial fibrosis are still not completely understood.
A growing body of evidence supports the key role of the renin-angiotensin-aldosterone system in the pathogenesis of AF (7). Recent evidences suggest atrial fibrosis is promoted by enhanced extracellular signal-regulated protein kinase (ERK) and angiotensin II type 1 receptor (AT1) activation in humans and animal models of AF (6-10). Progressive interstitial changes provoked by mitogen-activated protein kinase (MAPK), coupled to the angiotensin II regulatory pathway, increase the risk of AF (7). These results suggest that the atrial renin-angiotensin system plays a major role in the development of the remodeling process in AF. Aldosterone, a well-known effector hormone of the renin-angiotensin system, has a more pro-fibrotic role than angiotensin II (11). Aldosterone causes atrial fibrosis independent of changes in wall stress or hypertension (12). Reil et al. recently demonstrated that aldosterone induces a substrate for AF with locally disturbed conduction, independent of increased atrial load and characterized by
atrial fibrosis and myocyte hypertrophy (13). Furthermore, previous experimental heart failure and AF models showed that aldosterone blockade reduces atrial fibrosis and suppresses AF (12, 14-16). Clinically,plasma aldosterone levels are raised in AF patients, and the level of
mineralocorticoid receptor (MR) is higher in the atria of AF patients (17, 18). A recent genetic analysis also showed that a specific polymorphism related to increased aldosterone synthase activity predisposes heart failure patients to AF (19). Moreover, in the RALES trial, patients randomized to spironolactone had a significant reduction of both pro-collagen I and III when compared with patients receiving placebo (20). Therefore, aldosterone may contribute mechanistically to the pathogenesis of AF-promoting structural remodeling. However, the precise molecular mechanisms and signaling pathways involved are unknown. The purpose of our study was two-fold: 1) to examine the effect of aldosterone with different concentrations at different time intervals on the expression of AT1, MR, MAPK signaling molecules, and
fibrosis-related molecules in cultured HL-1 atrial myocytes ; 2) to determine the involvement of AT1/MR activity and MAPK signaling pathways in the mechanism of aldosterone-induced atrial fibrosis.
Methods
This experimental protocol was approved by the Institutional Animal Care and Use
Committee of the Chung Shan Medical University and complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 3040-2, revised 1999).
HL-1 Cell Culture
The HL-1 cell line derived from adult mouse atria was obtained from National Taiwan University Hospital, Taipei (through the courtesy of Dr. Chia-Ti Tsai). The HL-1 cells were cultured essentially as described by Claycomb et al (21). Briefly, cells were maintained in
Claycomb medium (JRH Biosciences, Lenexa, KS, USA) supplemented with 2 mM L-glutamine (Gibco, Grand Island, NY, USA), 0.1 mM norepinephrine (Sigma Chemical, St. Louis, MO, USA), 10% fetal bovine serum (FBS; JRH Biosciences), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in 5% CO2 at 37°C and 95% humidity. They were grown in 10-cm plates (dishes) coated with 5μg/ml fibronectin (Sigma) and 0.02% gelatin (Sigma) and until cells reached confluence. The cells were passaged every 3 days by trypsination (trypsin/EDTA, Invitrogen, Carlsbad, CA,USA), followed by addition of soybean trypsin inhibitor (Sigma). The initial plating density was in the range of 0.5−1.4 x 103 cells/mm2. Dishes with an initial plating density higher than 1.4 x 103 cells/mm2 are quickly overpopulated and the cells become
unhealthy. The cells used for experiments were starved in Claycomb medium without FBS 48 hours after seeding or until the cultures had reached approximately 80% confluence (22). HL-1 cells were plated in 35-mm culture dishes at a density of 1×106 cell/dish. Prior to experiments, cells were left in serum-free medium for 24 hours, and subsequently stimulated with aldosterone or different inhibitors. This was followed by protein isolation.
Drug Treatments
After serum starvation for 24 hours, cells were treated with aldosterone (10−5, 10−6, 10−8, 10−10, 10−12M) and subsequently harvested after 0, 1, 3, 6, 12 and 24 hours. Cells were
pre-treated with the following drugs as indicated:
The stock solution of Aldo, aldosterone powder ([1 mg/ml]; Fluka, Buchs, Switzerland) was prepared by addition of DMSO (Sigma), vortexing until dissolved, and storing at −20°C until use.
Aldosterone treatment was carried out by adding the stock solution (10 mM in DMSO) diluted in minimal medium of the apical and basolateral solutions to the above concentrations.
The stock solution of Epl, eplerenone-A (10 mM; Tocris, Avonmouth, Bristol, UK), and Los,
losartan A (1 mM; Fluka, Buchs, Switzerland), were prepared in DMSO according to the instructions provided with the product and diluted over 1 × 103-fold with medium immediately before use.
Protein Extracts and Western Blot Analysis
Using Western blotting, we investigated the effects of aldosterone with or without varied blockers on the expression of MR/AT1 levels, phosphorylation of different MAP kinases, and fibrosis-related proteins (TGF-β, α-SMA, COL1A and COL3A) in cultured HL-1 atrial cells.
Total protein was extracted from cells after culture under several experimental conditions using a Pro-prep protein extraction solution (iNtRON. Biotechnology, Seoul, Republic of Korea).
The homogenates were centrifuged at 15,000X g for 20 min at 4°C and stored at –20°C. Protein concentrations were determined using the DC Protein Assay kit (BioRad Laboratories, Inc., Hercules, CA,USA) and 50 μg of soluble protein per sample was loaded onto 8% sodium dodecylsulfate (SDS) polyacrylamide gels, subjected to electrophoresis (SDS-PAGE), and electrotransferred to polyvinylidene difluoridep (PVDF) membranes (Millipore, Bedford, MA, USA).
The membranes were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with either antibody against MR (Abcam, Cambridge, MA, USA), α-SMA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TGF-β1 (Geneway Biotech, San Diego, CA, USA), COL1A (Abcam), p38α, p38 MAPK(pT180/pY182), pan-JNK/SAPK,
JNK/SAPK(pT183/pY185), ERK, or ERK (pT201/pY204) (BD Biosciences, San Jose, CA, USA) at 4°C for overnight. A duplicate membrane was probed with anti-β-Actin (Geneway). The
membranes were then incubated with a species-specific horseradish peroxidase-labeled secondary antibody (BD Biosciences: 1:5000) for 1 hour at 37°C. Between each step, the
membranes were thoroughly washed with Tris buffered saline Tween (TBST). Peroxidase activity was detected on ImageQuant LAS 4000 mini biomolecular imager using ECL reagents (Millipore, Billerica, MA, USA). For quantification purposes, blots were quantified using a luminescent imager (LAS-1000 Image Analyzer, Fujifilm, Berlin, Germany) and FluorChem image software (Alpha Innotech, San Leandro, CA, USA).
Removal of primary and secondary antibodies was carried out at 50°C for 30 minutes in stripping buffer (100mM β-mercapthoethanol, 2% SDS, 62.5 mM Tris-HCl [pH 6.7]). After removal of antibodies, nonspecific interactions were re-blocked by incubating in TBST, 3% BSA for 1 hour before re-probing with primary antibody.
Immunohistochemical Analysis
For immunofluorescence, HL-1 cells grown on gelatin-fibronectin coated coverslips were serum starved for 24 hours, stimulated by 1 μM aldosterone under various conditions, fixed by 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized in 0.5%
Triton-X100 for 10 min, blocked with 1% BSA in PBS for 1 hour at room temperature, stained 2 hours at room temperature with antibody against MR (Abcam), α-SMA (Santa Cruz
Biotechnology), TGF-β1 (Geneway), COL1A (Abcam) and COL3A (Santa Cruz Biotechnology) all diluted 1:100 dilution in 1% BSA in PBS, washed with PBS twice, and incubated for an additional 60 min at room temperature with goat anti-mouse IgG or goat anti-rabbit IgG
conjugate with fluoresce in isothiocyanate (FITC) at 1:200 dilution in 1% BSA in PBS. Images were acquired with a DP72 digital camera and DP2-BSW microscope digital camera software.
As a negative control, HL-1 cells were incubated with the secondary antibody alone. To measure staining intensity, digital images were sectioned into basal and apical compartments and density of the immunofluorescent marker staining was performed using the pixel intensity routine from
Image J software (National Institutes of Health, Bethesda, MD, USA). Uniform microscope and laser settings were used for each experimental condition. Pixel intensity changes are expressed as a percent increase or decrease, and the magnitude of these changes was statistically evaluated.
Statistical Analysis
All data are expressed as mean ± SEM of at least three independent experiments. Data were compared using Student’s t-test for two-group data, and analysis of variance with posthoc t-test and corrections for P values was used for more than two group data. Paired t-test was used for comparison of protein level before and after aldosterone or the blockers. The P-values indicating statistical significance are given in the respective figure caption. A P value <0.05 was considered statistically significant.
Results Effect of Aldosterone on MR and AT1 Expression
To evaluate the effect of aldosterone on atrial renin-angiotensin activity, we exposed HL-1 cells to aldosterone at different concentration (10−5, 10−6, 10−8, 10−10, 10−12M) and for different time periods (1, 3, 6, 12 and 24 hours), and examined the protein expression of MR and AT1. As shown in Figure 1, aldosterone dose-dependently increased MR protein expression; aldosterone concentrations as low as 10−8 M induced a significant increase, and 24 hours of incubation with the highest concentration (10−5 M) induced a 2.57-fold increase (P<0.01). A significant increase in MR expression was seen after 1 hour exposure. Aldosterone induced an increase in AT1 protein expression in a concentration-dependent manner (Figure 1), with the maximal response ( 2.07-fold increase) reached at 10−8 M after 12 hours. A significant increase in AT1 expression was also seen after 1 hour exposure.
Subsequently, we treated HL-1 cells with a relatively high concentration (10−6 M) of
aldosterone, because plasma aldosterone level as high as 10−7 M has been reported in individuals with congestive heart failure.23 Aldosterone significantly and time-dependently increased MR protein levels, reaching a 2-fold increase after 24 hour (Figure 2A) and significantly increased AT1 protein levels at all times (P<0.01), reaching a maximal (1.76-fold) increase after 6 hours.
Aldosterone (10−6M) had no effect on the expression of glucocorticoid receptor.
Effects of Aldosterone on Fibrotic Markers Expression and MAPK Phosphorylation
To evaluate the effect of aldosterone on atrial fibrosis-related signaling pathway and proteins, we exposed HL-1 cells to aldosterone (10−6 M) and examined the time-dependency of the aldosterone effect on the expression of profibrotic proteins, and the activation of ERK, c-Jun N-terminal kinase (JNK), and p38MAPK. As shown in Figure 2B, the expression of COL1A and COL3A proteins (the major cardiac fibrotic markers) was significantly increased up to 1.55-fold and 1.37-fold (P<0.01), respectively. TGF-β1 (a critical profibrotic factor) and α-SMA (a trans-differentiation marker) were also up-regulated 1.38-fold and 1.44-fold (P<0.01),
respectively. Aldosterone significantly increased ERK, JNK, and p38MAPK phosphorylation (P<0.01) in a time-dependent manner (Figure 2C). Western blotting showed that JNK and p38 MAPK phosphorylation was induced within 1 hour and persisted for 24 hours, reaching a maximal (≈ 2-fold) at 24 hours. ERK phosphorylation was increased by 38% (P<0.01) at 12 hours and by 116% (P<0.01) at 24 hours.
Aldosterone-induced Fibrotic Cascade via MR Coupled to Functional AT1
To identify whether MR is involved in the aldosterone-induced fibrotic cascade and signaling, HL-1 cells were exposed to aldosterone (10−6 M) with or without pre-exposure to eplerenone (10−12 M and 10−10 M), a highly selective MR antagonist, to determine the expression
of MR and AT1, fibrosis marker proteins and ERK and JNK phosphorylation. As shown in Figure 3A, pre-treatment with eplerenone significantly inhibited the aldosterone-induced
increase in MR expression but significantly augmented the aldosterone-induced increase in AT1 expression. Eplerenone (10−10 M) significantly reduced COL1A level and abolished
aldosterone-induced phosphorylation of ERK and JNK, and eplerenone (10−12 M and 10−10 M) significantly reduced COL3A, TGF-β1 and α-SMA levels.
To investigate the effect of AT1 on the aldosterone-induced fibrotic cascade and signaling, HL-1 cells were exposed to aldosterone (10−6 M) with or without pre-exposure to the AT1 blocker losartan (10−12M and 10−10M). As shown in Figure 3B, losartan (10−10M) indeed abolished the aldosterone–induced increases in MR expression and COL1A, COL3A, TGF-β1 and α-SMA levels to those comparable to the negative control levels (vehicle in the absence of aldosterone and losartan), and abolished ERK but not JNK phosphorylation. Losartan (10−12 M) also significantly reduced aldosterone-induced TGF-β1 and α-SMA levels (P<0.05). These results implicated AT1 in some of the signaling pathways induced by aldosterone.
Taken together, these results suggested that activation of MAPK signaling and fibrotic marker proteins in aldosterone-treated HL-1 cells is tightly regulated by cross-talk between AT1 activation and MR activation in response to aldosterone. Our data support the critical role of elevated MR activity in the aldosterone-mediated increase in fibrosis marker proteins and activation of MAPK signaling. The response of aldosterone also required the presence of activated AT1. The amelioration by losartan of aldosterone-induced atrial fibrosis seems to be partially attributable to the attenuation of aldosterone-induced MR expression.
Aldosterone-induced Fibrotic Response via MAPK-Dependent Pathways
To further investigate whether aldosterone-induced collagen synthesis is mediated by the
MAPK signaling pathway, HL-1 cells were pre-incubated with U0126 (a specific MAPKK/MEK inhibitor of ERK activation) and SP600125 (an inhibitor of the JNK phosphorylation pathway) to examine the expression of MR, AT1, and fibrotic marker proteins, and pretreated cells were compared to those not pre-treated. As shown in Figure 4, U0126 (10−5 M) and SP600125 (10−5 M) prevented the aldosterone–induced increase in COL1A, COL3A, TGF-β1 and α-SMA levels, indicating that the aldosterone-induced fibrotic response is mediated by MAPK signaling pathway. Of particular interest in our data is that pre-treatment with MAPK pathway inhibitors abolished alosterone-stimulated MR and AT1 expression. This negative feedback effect of MAPK signaling inhibitors on MR expression lends further support to the notion that elevated MR activity plays a central role in promoting the aldosterone-induced fibrotic response mediated by the MAPK signaling pathway.
Immunohistochemical Analysis
We used immunofluorescence analysis to compared the relative density of fibrosis marker proteins in aldosterone-treated HL-1 cells with or without pre-treatment with blockers to MR (eplerenone), AT1 (losartan), or MAPK signaling (U0126 or SP600125). As shown in Figure 5, the densities were significantly reduced in all cells pre-treated by various blockers compared to those treated by aldosterone only. As noted previously, the increase in MR expression stimulated by aldosterone could be abolished in all the HL-1 cells by pre-treatment with the above blockers.
Taken together, our results suggested that MR/AT1 and the MAPK signaling pathway interact in a mutually cooperative manner to maintain the functional integrity of both AT1 and MR
necessary for the aldosterone-induced atrial fibrotic response.
Discussion
The major findings of our study on cultured HL-1 cardiomyocytes are that (1) aldosterone
treatment upregulates AT1 expression (positive feedback) independent of angiotensin II
production by atrial cells; (2) elevated MR activity plays a critical role in aldosterone-mediated activation of the MAPK signaling pathway and atrial fibrotic response; and (3) for the first time to our knowledge, treatment with AT1 blocker (losartan) and MAPK signaling inhibitors (U0126 or SP600125) downregualtes MR expression induced by aldosterone (negative feedback),
thereby preventing aldosterone-induced atrial fibrotic reactions. Our study supports the idea that interaction between MR/AT1 and the MAPK pathways produces positive feedback that is important in atrial fibrosis development.
Profibrotic Effects of Aldosterone Require Functional MR and AT1 Coupling
Aldosterone has been shown to upregulate ventricular and vascular AT1 and angiotensin converting enzyme expression, indicating the deleterious cardiovascular effects of aldosterone possibly mediated by activation of the renin-angiotensin system (24-27). The
aldosterone-mediated activation of AT1 in the absence of local angiotensin II production also represents an example of receptor transactivation by MR. Previous studies have shown that transactivation of MR is a critical aspect of angiotensin II-mediated cardiac or vascular signaling (28, 29). MR blockade downregulates angiotensin II-mediated signaling and reduces angiotensin II-induced end-organ damage, indicating that the effects of angiotensin II and MR activation are additive. On the other hand, the reciprocal interaction between aldosterone and AT1 has also been reported. Aldosterone upregulates the expression of angiotensin-converting enzyme and AT1, and potentiates angiotensin II-stimulated intracellular signaling and proliferation in cardiovascular diseases (24-27). Furthermore, Robert et al. demonstrated that AT1 blockade significantly prevents the aldosterone-induced cardiac collagen expression and cardiac fibrosis in vivo (26). Thus, aldosterone-induced cardiac fibrosis seems to be partially attributable to the
cross-talk between aldosterone and AT1, either directly or indirectly. However, mechanisms underlying this interaction remain unclear. Spironolactone, an MR antagonist, has been shown to suppress the upregulation of angiotensin converting enzyme and AT1 by aldosterone, suggesting that MR is part of a positive feedback loop for local control of the renin-angiotensin system (26, 27, 30). These results suggest that aldosterone-induced cardiac and vascular damage depends in part on activation of an angiotensin II-AT1-mediated pathway. By contrast, our study showed that pre-treatment with eplerenone (a specific MR blockade) significant enhanced
aldosterone-induced AT1 upregulation and reduced MR expression in cultured HL-1 atrial cells.
Discrepancies between our study and prior studies may be explained in part by the use of different cell types (atrial versus ventricular or vascular smooth muscle cells). Previous experiments have shown that aldosterone interacts with angiotesin II-AT1 signaling via an MR-independent mechanism or is mediated by a plasma membrane receptor distinct from the classical cytosolic MR first proposed by Wehling et al (31). Chai et al. showed that aldosterone potentiated the vasoconstrictor effect of angiotensin II in coronary arteries, and that this effect was not blocked by spironolactone (32). Yamada et al. further demonstrated that the
vasoconstrictor effect of aldosterone is nongenomic and independent of classical MR and is not blocked by spironolactone but by AT1 blockers (33). Thus, it seemed reasonable to assume that aldosterone could transactivate AT1 independent of MR in HL-1 atrial myocytes. Our results also indicated that the aldosterone-induced activation of MAPK signaling and fibrotic marker
proteins is directly mediated by MR and not by activation of AT1. However, we also showed that pre-treatment with the AT1 inhibitor, losartan, could abolish the increase in MR expression induced by aldosterone in parallel with reduction of ERK phosphorylation and
fibrosis-associated proteins expression, indicating that the effects of aldosterone depend on AT1
activation. In vitro studies of vascular smooth cells exposed to aldosterone demonstrated increased production of reactive oxygen species, transactivation of the epidermal growth factor receptor, and activation of ERK and JNK signaling, whereas AT1 blockade inhibited these effects (28). Moreover, by using small interference RNA to knockdown AT1, Lemarié et al.
demonstrated that aldosterone-induced activation of signaling pathways requires AT1 activity (34). We not only found that MR and AT1 cross talk, but most importantly, that the activation of MR by aldosterone requires a functional AT1. The present report to our knowledge is the first to demonstrate that AT1 blockade abrogated the profibrotic effect of aldosterone is probably mediated by the attenuation of aldosterone-induced MR expression. Our results shed new light on the mechanism of cross-talk between AT1 activation and MR activation. The activation of the ERK MAPK signaling pathways and profibrotic changes in response to aldosterone in cultured HL-1 atrial myocytes requires a functional AT1 coupled to elevated MR activity. These results were confirmed by pharmacological blockade of AT1 and MR, demonstrating the clinical relevance of the results from the present study.
Elevated MR Activity is Prerequisite in Aldosterone-Induced Atrial Fibrotic Response
AF is associated with substantial structural changes in atrial tissue that, to a large extent, depend on increased activity of the renin-angiotensin-aldosterone system (2, 7). Atrial fibrosis is the hallmark of structural modeling. Aldosterone is increasingly recognized to be involved in the process of atrial remodeling (12-20). Milliez et al.demonstrated that only spironolactone rather than angiotensin-converting enzyme inhibitor induced a significant decrease in atrial fibrosis in rats with heart failure following myocardial infarction, suggesting a distinct regulatory pathway and profile of atrial remodeling (14). Although previous studies support that atrial tissue is a target of aldosterone, MR-mediated signaling in atrial cells has not been systematically explored.
MR is the nuclear receptor of aldosterone and is expressed in both cardiac myocytes and fibroblasts. Eplerenone used in our study is considered to be a more selective MR antagonist without the side effects of spironolactone, including gynecomastia and breast pain, because of its low affinity for androgen and progesterone receptors. In experiments, eplerenone has been shown not merely to prevent but also reverse established cardiac fibrosis, and to suppress AF occurrence (15, 35, 36). A recent clinical trial also confirmed the added reduction in new-onset AF when eplerenone was used in patients with systolic heart failure and mild symptoms (37).
We demonstrated that aldosterone-treated cells increase their expression of MR and AT1 proteins in a concentration-dependent and time-dependent manner. Aldosterone also
time-dependently induced activation of ERK, JNK, and p38MAPK signaling and expression of fibrosis marker proteins (COL1A, COL3A, TGF-β1 and α-SMA). The significant upregulation of MR, AT1, JNK, p38MAPK, and COL1A by aldosterone stimulation occurred within 1 hour.
Aldosterone can exert pro-inflammatory vascular effects via MR- and ERK1/2
MAPK-dependent mechanisms (38). In accordance with previous studies showing that increased MR expression and ERK and JNK MAPK activity are involved in the process of
aldosterone-induced profibrotic response, MR blockade by eplerenone and inhibition of MAPK signaling by U0126 or SP600125 reduced expression of fibrotic marker proteins in HL-1 cells to negative control levels (39). We also revealed that aldosterone induced activation of ERK and JNK MAPK through classical MR. Recent evidence indicates aldosterone has both slow, genomic and rapid, non-genomic effects, which typically observed within minutes after aldosterone stimulation and include activation of MAPK signals. Aldosterone-mediated
pathologies, such as inflammation, remodeling, and endothelial dysfunction, are characterized by activation and cross-talk of genomic effects with rapid signaling pathways (28, 38, 40).Our
results do not show to what extent genomic and nongenomic mechanisms contribute to the observed effects. Thus, MR-mediated genomic actions or MR-mediated nongenomic MAPK signaling may both play a significant role in aldoserone-induced profibrotic marker expression in our cell model.
The most promising finding of the present study is that pre-treatment of cells with blockers to either receptor (losartan, eplerenone) or MAPK signaling (U0126 or SP600125) reduced levels of aldosterone-induced MR and fibrosis-related proteins, as demonstrated by Western blot and immunofluorescence analysis. Our study findings favor a growth-promoting role of MR in AF. Jaffe and Mendelsohn demonstrated that spironolactone inhibited angiotensin II-mediated MR-dependent gene expression in vascular smooth muscle cells, and highlighted a specific role for MR in regulating angiotensin II and aldosterone gene transcription (41). Increased
constitutive MR signaling has been shown to promote age-associated vascular inflammation (42).
Moreover, Tsai et al. demonstrated that the expression of the MR is increased in patients with AF compared to patients with sinus rhythm, indicating that MR expression is the major factor
determining the effects of serum aldosterone on the atrial tissue (18). Our study further illustrated that blocking AT1 or MAPK signal pathways has a negative feedback effect (i.e., inhibits
aldosterone-induced MR expression and subsequent events). Fraccarollo et al. recently demonstrated that deletion of cardiomyocyte MR improves infarct healing and prevents progressive adverse cardiac remodeling, highlighting a central role for cardiomyocyte MR in post-infarction healing and remodeling (43). The present report to our knowledge is the first to demonstrate that downregulation of MR expression seems to be the final common pathway to abolish aldosteone-mediated profibrotic effects. We demonstrate here for the first time that increased MR activity is prerequisite in the development of the atrial fibrotic response to
aldosterone. In the absence of AT1 or MR blockade, increased tissue levels of aldosterone will upregulate MR activity and feedback loops in the local renin-angiotensin system (e.g., AT1 expression), resulting in a number of vicious cycles. In vivo, angiotensin II and aldosterone may modulate signaling pathways and cardiovascular function either synergistically or via a cross-talk between their receptors or their respective messenger systems. In addition, recent studies have showed that angiotensin II has several pro-inflammatory properties, and the converse is also true with inflammatory stimulating angiotension II production. Thus, angiotensin II-AT1,
aldosterone-MR and profibrotic pathways may interact in a mutually cooperative manner to produce positive feedback that is important in fibrosis development.
Clinical Implication
Pathophysiological synergistic effects between angiotensin II and aldosterone have been described on cardiovascular tissues (8, 28, 38).From a clinical viewpoint, our results provide evidence for a role of MR and aldosteorne signaling in the development of atrial fibrosis,
expanding the mechanistic explanation of the complex interactions between the activation of MR, AT1 and their signals. Our evidence from this experimental in vitro study supports the beneficial role of MR blockade in conjunction with AT1 blockade or angiotensin-converting enzyme inhibitor in the prevention of AF occurrence. Clinical evidence, however, is scarce and further research is clearly warranted. Of note, EMPHASIS-HF trial has shown that eplerenone amplifies the benefits of angiotensin receptor blockers in reducing new-onset AF in patients with systolic heart failure and mild symptoms (37).
Limitation
The collagen mRNA or protein expression is often different from collagen volume fraction because collagen synthesis depends on the time course of the cardiovascular disease and the
underlying cardiovascular disease itself. Therefore, whether results in culture HL-1 cells can be readily extrapolated to atrial tissues impaired by different underlying cardiovascular diseases is unknown. In fact, it is not known whether aldosterone signaling differs between subjects with underlying chronic conditions such as ischemic cardiomyopathy, dilated cardiomyopathy, hypertension, and so on. It is also unclear whether aldosterone signaling changes with the extent of atrial remodeling. Furthermore, the role of MR blockade in different types and stages of atrial remodeling remains to be determined.
Conclusions
Aldosterone is a pathophysiological mediator of atrial fibrosis. Aldosterone mediates its fibrotic effects on HL-1 atrial cells via MR and the MAPK intracellular signaling cascade. Our study provides evidence that increased MR signaling has a central role in promoting atrial
fibrotic responses, and the mutual feedback between AT1-MR and MAPK signaling activation is important for the development of these responses.
Acknowledgments
The authors thank Dr. CT Tsai, Department of Internal Medicine, National Taiwan
University Hospital, for the kind provision of the HL-1 cell line derived from adult mouse atria.
Disclosures None.
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Figure Legends
Figure 1. Effect of aldosterone on MR and AT1 expression. HL-1 cells were incubated with aldosterone at the indicated concentrations for the indicated times. MR and AT1 expressions were measured in cellular homogenates by Western blot analysis. Western blots contained 25 μg total protein/lane and were probed with the indicated antibody. All data were normalized to β-actin shown as a control for equal loading and blotting. Aldosterone concentration-dependently increased MR and AT1 protein expression in HL-1 cells with maximum response at 10−5 M and 10−8 M, respectively. The bar graph represents the densitometric quantification of MR and AT1 immunoreactivity, expressed as fold increase over the untreated control level at 0 hour. Values are mean±SEM (n=3, *P<0.05, **P<0.01 versus vehicle).
Figure 2. Time-dependent effect of aldosterone on indicated protein expression. HL-1 cells were incubated with aldosterone (10−6 M) for the indicated times. The top panels in A through C show representative Western blots of the respective proteins. The bottom panels in A through C are bar graphs showing band intensities after densitometric quantification of the indicated protein bands. Each column showing the fold increase over control is the mean from three independent experiments. Panel A: Aldosterone time-dependently increased MR and AT1 protein expression in HL-1 cells (maximum reached at 24 and 6 hours, respectively). Panel B: Aldosterone
time-dependently increased TGF-β1, α-SMA, COL1A and COL3A protein levels in HL-1 cells (maximum reached at 24 hours). Panel C: To detect activation of MAP kinases,
phospho-specific antibodies that selectively recognize the active forms of p38MAPK, ERK1/2 and JNK were used. Antibodies that bind to either form of p38MAPK, ERK1/2, and JNK were used to ensure equal loading of the samples. Phosphorylated and total p38MAPK, ERK, or JNK
were detected by immunoblot. Aldosterone time-dependently increased p38MAPK, ERK1/2 and JNK protein levels (maximum reached at 24 hours). The data are mean ± SEM (n=3, *P<0.05,
**P<0.01 vs. vehicle).
Figure 3. Panel A: Effect of eplerenone on aldosterone-stimulation of the indicated protein expressions. The HL-1 cells were pre-incubated with eplerenone (Epl; 10−10 or 10−12 M) for 30 min and then stimulated with aldosterone (10−6 M) for 24 hours. Pre-treatment with eplerenone significantly inhibited aldosterone-induced increases in MR, COL1A, COL3A, TGF-β1, and α-SMA expression, and phosphorylation of ERK and JNK. By contrast, eplerenone significantly augmented the aldosterone-induced increase in AT1 expression. Panel B: Effect of losartan on aldosterone-stimulation of the indicated protein expressions. The HL-1 cells were
pre-incubated with losartan (Los; 10−10 or 10−12 M) for 30min and then stimulated with aldosterone (10−6 M) for 24 hours. Pre-treatment with losartan significantly inhibited
aldosterone-induced increases in MR, COL1A, COL3A, TGF-β1, and α-SMA expression, and phosphorylation of ERK. The data are means ± SEM (n=3, *P<0.05, **P<0.01 vs. control) from three independent experiments.
Figure 4. Effects of ERK or JNK pathway blockade on aldosterone-stimulation of the indicated protein expressions. After 20 hours in serum-free medium, cells were pre-incubated with U0126 (10−5 M) or SP600125 (10−5 M) or DMSO as a vehicle for 2 hour and then treated with 10−6 M aldosterone for 24 hours. Pre-treatment with U0126 and SP600125 indeed abolished ERK and JNK activation, respectively, thereby significantly preventing aldosterone–induced increase of the levels of COL1A, COL3A, TGF-β1, and α-SMA. Notably, pre-incubating with MAPK pathway inhibitors abolished aldosteron-stimulated MR and AT1 expression. Data are mean ± SEM (n=3, *P<0.05, **P<0.01 vs. control) from three independent experiments.
Figure 5. Immunohistochemistry assay of aldosterone-treated HL-1 cells. Cells were pre-treated with inhibitors as indicated (Epl, Los, U0126 and SP600125) or vehicle, and then stimulated with aldosterone (10−6 M) for 24 hours. HL-1 cells were fixed and subjected to immunostaining with anti-COL1A, anti-COL3A, anti-TGF-β1, or anti-α-SMA antibody. Pixel intensity of the cells displaying fluoresces accumulation was measured. All indicated fibrotic marker protein densities were significantly reduced in all the cells pre-treated by various blockers compared to those treated by aldosterone only. Values are the arithmetic means of at least three independent experiments. The percentage of fluoresces cells was calculated per condition as described in Methods (n >100, *P<0.01 vs. control).
國科會補助計畫衍生研發成果推廣資料表
日期:2012/10/15
國科會補助計畫
計畫名稱: C急性反應蛋白在HL1細胞株及新生心房纖維母細胞向上調控血管收縮素II第 一型接受體
計畫主持人: 翁國昌
計畫編號: 99-2314-B-040-020-MY2 學門領域: 心胸內科
無研發成果推廣資料
99 年度專題研究計畫研究成果彙整表
計畫主持人：翁國昌 計畫編號：99-2314-B-040-020-MY2
計畫名稱：C 急性反應蛋白在 HL1 細胞株及新生心房纖維母細胞向上調控血管收縮素 II 第一型接受體 量化
成果項目 實際已達成
數（被接受 或已發表）
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達成數)
本計畫實 際貢獻百
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件數 0 0 100% 件
技術移轉
權利金 0 0 100% 千元
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（本國籍）
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期刊論文 0 0 100%
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研討會論文 0 0 100%
論文著作 篇
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申請中件數 0 0 100%
專利 已獲得件數 0 0 100% 件
件數 0 0 100% 件
技術移轉
權利金 0 0 100% 千元
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博士生 0 0 100%
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（外國籍）
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(無法以量化表達之成
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國科會補助專題研究計畫成果報告自評表
請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價 值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）、是否適 合在學術期刊發表或申請專利、主要發現或其他有關價值等，作一綜合評估。
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心房顫動是臨床最常見的心律不整。在一般人群的總患病率在 0.4％，隨著年齡增加，心 房顫動有逐漸增加的趨勢，在 75 歲以上人群可達 10％。心房顫動會明顯增加腦中風及心 臟衰竭甚至死亡的機會，因此心房顫動是消耗社會成本很重的一種心律不整的疾病。近年 來有許多研究指出腎素-血管收縮素-留鹽激素系統 (renin-angiotensin-aldosterone system, RAAS)與發炎反應及 AF 有潛在的關連性。最近的研究發現，在老鼠體內，心房壓 力上升時，會促使心房血管收縮素上升，進而造成心房結構重構，使心房纖維化(AF)及心 房擴大。心房纖維顫動的發生和腎素-血管張力素系統有很大的關係，血管張力素可使得 心房纖維顫動更容易發生及維持。當血管收縮素結合在血管收縮素 II 第一型接受體時，
會促使血管收縮使血壓上升，並促進腎上腺增加留鹽激素的產生和分泌，留鹽激素亦會調 控血管收縮素 II 第一型接受體的表現。本研究的目的，主要在於留鹽激素和心房纖維顫 動發生機轉的關係。探討 Aldosterone 是否造成心肌細胞內 AT1； mineralocorticoid receptors (MR)及纖維化指標分子等蛋白表現量的改變，進而影響心臟之功能。在本研究 報告，我們利用細胞模式，來探討留鹽激素在促進心房纖維顫動之可能機制。研究結果顯 示，在培養 HL-1 心房細胞時，加入留鹽激素可以促使 MR、AT1 及纖維化指標分子蛋白表 現增加，並且觀察 MAPK 活化的表現；在加入留鹽激素拮抗劑 eplerenone 或血管收縮素 第一型受體阻斷劑 losartan 會抑制留鹽激素促使纖維化指標分子（COL1A, COL3A,
扮演重要的角色，其機轉和 MAPK 之相關訊息傳導路徑有關。
