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STAT Pathway Mediates the Induction of Visfatin in Angiotensin II-Induced Cardiomyocyte Hypertrophy
BASIC INVESTIGATION
Angiotensin II Type-1 Receptor-JAK/STAT Pathway Mediates the Induction of Visfatin in Angiotensin II-Induced Cardiomyocyte Hypertrophy Liang Chang, MM, Rong Yang, MM, Mei Wang, MM, Jinming Liu, MM, Yaling Wang, MM, Hui Zhang, MM and Yongjun Li, PhD
Abstract: Introduction: The new adipocytokine visfatin is closely associated with the cardiovascular diseases, and expression of visfatin is elevated in the heart failure patients. However, at the cellular level, little work has been done on visfatin expression in the cardiomyocyte hypertrophy. Here, the authors investigated the expression and mechanisms of visfatin in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy in vitro by means of the cultured neonatal rat cardiomyocytes. Methods: After primary culture of 2- to 3-day-old Sprague– Dawley rat cardiomyocytes and cardiac fibroblasts, cardiomyocytes were pretreated with Ang II. Ang II type-1 receptor (AT1-R) antagonist telmisartan and Ang II type-2 receptor antagonist PD123319 were used to block effects of Ang II. These inhibitors used for the AT1-R pathway determination included SP600125, AG490 and U0126. Cell viability was examined using the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The expression of visfatin was examined by means of reverse transcription-polymerase chain reaction and Western blot. The expression of brain natriuretic peptide was examined through western-blot analysis. Results: Visfatin was found expressed in cardiomyocytes as well as cardiac fibroblasts, and there was no significant difference at the mRNA and protein levels of visfatin. Ang II treatment induced the increased expression of visfatin and brain natriuretic peptide in a dose- and time-dependent manner in cardiomyocytes, and pretreatment with AT1-R antagonist telmisartan completely blocked Ang II-induced visfatin expression increasement. The increased visfatin expression was also blocked by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway inhibitor AG490. Conclusion: Visfatin expression was increased mainly through the AT1-R-JAK/STAT pathway in the process of Ang IIinduced cardiomyocyte hypertrophy. Key Indexing Terms: Visfatin; Myocyte; Hypertrophy; Angiotensin II. [Am J Med Sci 2012;343(3):220–226.]
T
he cardiomyocyte hypertrophy is one of the main ways in which the cardiomyocytes respond to the mechanical and neurohormonal stimuli. It is well known that sustained increasement against the systolic or diastolic wall stress will lead to the cardiomyocyte hypertrophy. Both in vitro and in vivo studies show that trophic responses to stretch (increases in diastolic wall stress) involve angiotensin II (Ang II).1 Ang II also exerts the direct growth-promoting effect on cardiac tissues, resulting in the cardiomyocyte hypertrophy and the mechanical dysfunction independent of pressure overload.2,3 Ang II activates several intracellular signal transduction pathways, and therefore the local increase in the Ang II concentration could significantly From the Department of Cardiology, The Second Hospital of Hebei Medical University; and The Hebei Institute of Cardiovascular and Cerebrovascular Diseases, Shijiazhuang, Hebei, China. Submitted March 3, 2011; accepted in revised form June 8, 2011. Correspondence: Yongjun Li, PhD, Shijiazhuang, Hebei 050000 (E-mail: [email protected]).
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contribute to the pathogenesis of cardiomyocyte hypertrophy, even in the absence of arterial hypertension.4 In addition, the cardiomyocyte hypertrophy is often accompanied by the increasement of protein synthesis and the overexpression of hypertrophic genes, such as brain natriuretic peptide (BNP).5,6 Normally, BNP is secreted mainly by the embryonic cells and adult ventricular myocytes. However, in the pathological cases, the ventricular myocytes transform from contraction type to synthesis type promoted by hypertrophy factors, and BNP gene expression increases. Hence, BNP has been recognized as the sign of cardiomyocyte hypertrophy.7 The adipose tissue is not only considered as the energy storage tissue but also a significant endocrine organ. Adipocytes secrete a series of vasoactive substances, named adipokines, such as leptin, tumor necrosis factor-␣, interleukin-6, resistin and adiponectin. Samal et al8 cloned a kind of protein from peripheral blood lymphocytes, which could promote the proliferation of pre-B cells, and therefore, they named it the pre-B-cell colony-enhancing factor (PBEF). Rongvaux et al9 demonstrated that PBEF was nicotinamide phosphoribosyltransferase, which was a cytosolic enzyme in nicotinamide adenine dinucleotide biosynthesis. Fukuhara et al10 reported a kind of adipocytokine, which was highly expressed in visceral fat, was called visfatin and corresponds to PBEF. However, recent studies contradict several previously reported results, showing that adipokines could be secreted and expressed not only in the adipose but also in the other organs. Meantime, tumor necrosis factor-␣, interleukins, adiponectin and leptin are synthesized and secreted not only in the adipose but also in the heart.11–15 The new adipokines visfatin can be expressed both in the visceral fat tissues and in the heart tissues.16 Visfatin is widely expressed in a variety of organs, which indicates its versatility. It is well known that visfatin plays an important role in the cardiovascular diseases.17 Visfatin is highly expressed in the lipid-rich macrophages and in patients with the acute coronary syndrome.18 Visfatin can promotes maturation of the vascular smooth muscle cells and contribute to the vascular remodeling.19 Plasma visfatin concentrations significantly increased in patients with heart failure.20 However, at the cellular level, little work has been done on visfatin expression in the cardiomyocyte hypertrophy. In addition, previous studies have found that visfatin was expressed in the heart, but so far, no any research focuses on expression of visfatin in the cardiac cells. Therefore, in this study, we examined expression of visfatin between cardiac fibroblasts and cardiomyocytes and investigated the expression of visfatin and its mechanisms in Ang II-induced cardiomyocyte hypertrophy in vitro by the cultured neonatal Sprague–Dawley rat cardiomyocytes.
The American Journal of the Medical Sciences • Volume 343, Number 3, March 2012
Visfatin Expression in Cardiac Hypertrophy
MATERIALS AND METHODS All experimental procedures in this study conformed to Hebei Province Experimental Animal Management Methods. No anesthetics were administered to avoid interferences with biochemical values. Preparation of Cardiomyocytes and Cardiac Fibroblasts and Cells Culture Ventricular cardiomyocytes were dissociated from the 2to 3-day-old neonatal Sprague–Dawley rats (The Laboratory Animal Center of Hebei Province, Shijiazhuang, China), preplated in 2 steps and plated onto 6 well microplates (Corning Costar, New York, NY) to yield confluent cardiomyocytes, as described previously.21 These cells were incubated in the culture medium with 10% fetal calf serum (Solarbio, Beijing, China) at 37°C in humidified air with 5% CO2. 5-bromo-2deoxyuridine (Santa Cruz Biotechnology, Santa Cruz, CA) 0.1 mM was routinely applied for 48 hours to inhibit cardiac fibroblasts growth.22 Forty-eight hours after seeding, the cardiomyocytes were cultured with the serum-free culture medium for another 24 hours, which were pretreated with Ang II (Sigma-Aldrich, St. Louis, MO). The neonatal rat cardiac fibroblasts were recovered from the preplating steps of the cardiomyocytes isolation and cultured in the maintenance media, which was the same as above. Upon proper confluence, the fibroblasts were split and passaged at a 1:2 ratio and used at passage one.23 Analysis of Cardiomyocytes Viability Effects of Ang II on cardiomyocytes viability were determined by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (HyClone, Logan City, UT). The cardiomyocytes were plated at a density of 1 ⫻ 105/mL in a 96-well plate. After 48 hours after seeding, the cardiomyocytes planted on 96-well plates (Corning Costar) were serum starved for another 24 hours before treatment with Ang II. Then, these cardiomyocytes were incubated with 20 L
of 5 mg/mL MTT solution for 4 hours at 37°C. Thereafter, 150 L of dimethyl sulphoxide (HyClone) was added to each well to dissolve the dye crystal formazan, and the plate was shaken for 10 minutes to make sure that all purple crystals were completely dissolved. The amount of MTT formazan was quantified by measuring the absorbance at 490 nm using a microplate reader. RNA Isolation and Reverse Transcription-polymerase Chain Reaction Analysis Total RNA was isolated from the cardiomyocytes and cardiac fibroblasts by TRIzol reagent (SBS, Beijing, China). The purity of RNA was assessed by measuring the absorbance at 260 nm. A specific 338-bp size fragment was amplified using the primers for examining visfatin gene expression (forward: 5⬘-ACTTTGAATGCCGTGAA-3⬘; reverse: 5⬘-AATCCAGTTGGTGAGCC-3⬘). Glyceraldehyde phosphate dehydrogenase expression was used as internal control (forward: 5⬘-GAGGCTCTCTTCCAGCCTTC-3⬘; reverse: 5⬘-AGGGTGTAAAAGCAGCTCA-3⬘), and the fragment of 380-bp size was amplified. The reverse transcription-polymerase chain reaction was run for 30 cycles under the following conditions. The DNA template was denaturated at 94°C for 60 seconds, specific annealing at 52°C for 60 seconds and 72°C for 60 seconds, with a final extension step at 72°C for 10 minutes. Amplification was linear under these conditions and was carried out in a Biometra T-gradient Thermoblock PCR System (Santa Cruz Biotechnology). All RT-PCRs for each gene were performed at the same time and with the same batch of Taq polymerase to reduce variations in RT-PCR efficiency. Band densities were quantified using a scanning densitometer coupled to scanning software Gel-Pro Analysizer 3.1 (Informax, Bethesda, MD). Western Blot Analysis The visfatin and BNP protein expression in the cardiomyocytes were examined using Western blot analysis. The cells were lysed with the radioimmunoprecipitation buffer (Sigma-
FIGURE 1. Cell morphology and visfatin expression in cardiac cells. (A) Cardiac fibroblasts 200⫻. (B) Cardiomyocytes 200⫻. Visfatin mRNA (C) and protein (D) expression in cardiac fibroblasts (1) and cardiomyocytes (2).
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FIGURE 2. Cell viability, visfatin and BNP expression in cardiomyocytes pretreated with different concentration of Ang II for 48 hours. (A) Analysis of cardiomyocytes viability with 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Visfatin mRNA (B) and protein (C) expression in cardiomyocytes: 1, control; 2, Ang II 10⫺8 mol/L; 3, Ang II 10⫺7 mol/L; 4, Ang II 10⫺6 mol/L; 5, Ang II 10⫺5 mol/L. (D) BNP protein expression in cardiomyocytes: 1, control; 2, Ang II 10⫺8 mol/L; 3, Ang II 10⫺7 mol/L; 4, Ang II 10⫺6 mol/L; 5, Ang II 10⫺5 mol/L. **P ⬍ 0.01; ANOVA for comparison against control group.
Aldrich). After centrifugation and quantification, the samples were boiled before use. For analysis, 80 g of each sample was loaded. Each sample were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto the polyvinylidene fluoride membrane (Millipore, Bedford, MA). The polyvinylidene fluoride membranes were then blocked by incubation with 5% skim milk (Becton, Dickinson and Company, Franklin, NJ) in 20 mM Tris 䡠 Cl, 150 mM NaCl, 0.05% Tween 20 tris-buffered solution (TBS) plus Tween 20 (TBS-T), pH 7.4, for 1 hour at room temperature. The membranes were then incubated with the primary antibody anti-PBEF (Santa Cruz Biotechnology; rabbit polyclonal 1:200 diluted in 5% TBS-T) and the primary antibody anti-BNP (Abcam, Cambridge, UK; rabbit polyclonal 1:500 diluted in 5% TBS-T) overnight at 4°C. These membranes were then washed thoroughly for 30 minutes at room temperature with TBST (0.1%) followed by incubation with the secondary antibody IRDye700DX and IRDye800CW (1:5000; Rockland, Gilbertsville, PA) (1:4000 dilution) for 1 hour at room temperature. The antibody complexes were visualized using chemiluminescence (Odyssey9120; LI-COR Corporation, Lincoln, NE). Band densities were measured using a scanning densitometer coupled to scanning software Gel-Pro Analysizer 3.1. These mem-
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branes were also reprobed with glyceraldehyde phosphate dehydrogenase (Santa Cruz Biotechnology) to determine the equal protein loading. Statistical Analysis Data were normalized for the housekeeping gene glyceraldehyde phosphate dehydrogenase and expressed as a mean ⫾ SD for 3 times experiments and analysis by t test and ANOVA, using SPSS 10.0 software. Differences were considered statistically significant when P ⬍ 0.05.
RESULTS Cell Morphology and Visfatin Expression in Cardiac Cells The cardiomyocytes (Figure lA) and cardiac fibroblasts (Figure lB) were observed under the inverted microscope (200⫻). As expected, PCR products amplified using the specific primers for visfatin showed a clear band at the predicted size of 338 bp both in the cardiomyocytes and cardiac fibroblasts (Figure 1C). Using Western blot analysis, visfatin protein expression was confirmed both in cardiomyocytes and cardiac fibroblasts (Figure 1D). As shown in Figure 1, however, there was no significant Volume 343, Number 3, March 2012
Visfatin Expression in Cardiac Hypertrophy
FIGURE 3. Cell viability, visfatin and BNP expression in cardiomyocytes pretreated with Ang II 10⫺6 mol/L for different times (6, 12, 24 and 48 hours). (A) Analysis of cardiomyocytes viability with 3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide. Visfatin mRNA (B) and protein (C) expression in cardiomyocytes: 1, 6 hours (a, control; b, Ang II); 2, 12 hours (a, control; b, Ang II); 3, 24 hours (a, control; b, Ang II); 4, 48 hours (a, control; b, Ang II). (D) BNP protein expression in cardiomyocytes: 1, control; 2, 6 hours; 3, 12 hours; 4, 24 hours; 5, 48 hours. **P ⬍ 0.01; ANOVA for comparison against control group.
difference in visfatin mRNA and protein expression between the cardiac fibroblasts and the cardiomyocytes. Ang II Induced an Increase in Cardiomyocytes Viability and the Expression of Visfatin and BNP The cardiomyocytes were treated with an increasing concentration of Ang II (10⫺8 mol/L, 10⫺7 mol/L, 10⫺6 mol/L and 10⫺5 mol/L) for 48 hours. The optical density (OD) values of MTT products of the cardiomyocytes pretreated with Ang II increased in a dose-dependent manner, peaking when the concentration of Ang II was 10⫺6 mo1/L, and it dropped significantly when the concentration of Ang II was 10⫺5 mo1/L (Figure 2A). At the mRNA level, visfatin expression was increased in a dose-dependent manner, which was the highest when the concentration of Ang II was 10⫺5 mo1/L, and it slightly lowered when the concentration of Ang II was 10⫺6 mo1/L, and there was no significant difference (Figure 2B). The same was for visfatin protein expression (Figure 2C). BNP protein expression increased in a dose-dependent manner, which was the highest when the concentration of Ang II was 10⫺6 mo1/L, and it significantly dropped when the concentration of Ang II was 10⫺5 mo1/L (Figure 2D). The cardiomyocytes were then treated with Ang II 10⫺6 mol/L at different times (6, 12, 24 and 48 hours). The OD values of MTT products of the cardiomyocytes were increased © 2012 Lippincott Williams & Wilkins
in a time-dependent manner, peaking at 12 hours, and it significantly decreased after 24 hours (Figure 3A). The cultured normal cardiomyocytes at different times (6, 12, 24 and 48 hours) were used as control groups. There was no difference at the mRNA and protein levels of visfatin at different periods between control groups. However, visfatin mRNA and protein expression were significantly higher than the corresponding control groups. Visfatin mRNA and protein expression were increased in a time-dependent manner, which were the highest at 24 hours, and significantly decreased after 48 hours (Figures 3B and 3C). BNP protein expression increased in a timedependent manner, which was the highest at 24 hours, and slightly decreased at 48 hours, but there was no significant difference between the 24-hour group and the 48-hour group (Figure 3D). Mechanisms of the Visfatin Expression Increasement in Ang II-Induced Cardiomyocyte Hypertrophy To investigate mechanisms of the visfatin expression increasement in the Ang II-treated cardiomyocytes, we blocked Ang II type-1 receptor (AT1-R) and Ang II type-2 receptor (AT2-R) activities by using telmisartan (10 mol/L) and PD123319 (10 mol/L), respectively. The cells were exposed to telmisartan and PD123319 30 minutes before the addition of AngII 10⫺6 mol/L to the medium and the cells were then
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FIGURE 4. Effects of Ang II receptor and AT1-R signalling inhibitors on visfatin expression. Visfatin mRNA (A) and protein (B) expression in cardiomyocytes treated with Ang II 10⫺6 mol/L for 24 hours in the presence of telmisartan (10 mol/L) or PD123319 (10 mol/L): 1, control; 2, Ang II; 3, telmisartan ⫹ Ang II; 4, PD123319 ⫹ Ang II. Visfatin protein (C) and mRNA (D) expression in cardiomyocytes treated with Ang II 10⫺6 mol/L for 24 hours in the presence of AG490 (20 mol/L), U0126 (20 mol/L) or SP600125 (20 mol/L). **P ⬍ 0.01; ANOVA for comparison against control group.
incubated in the presence of both drugs for a further 24 hours. We found that visfatin mRNA and protein expression were the highest in the PD123319 group, followed by the Ang II group, and at the same time, there was no significant difference between the telmisartan and the control groups (Figures 4A and 4B). These inhibitors used for the AT1-R pathway determination were SP600125 [c-Jun NH 2-terminal kinase (JNK)] (10 mol/L), U0126 [extracellular signal-regulated kinase (ERK1/ 2)] (10 mol/L) and AG490 [Janus kinase (JAK)] (10 mol/L). The cells were exposed to inhibitors 30 minutes before the addition of Ang II 10⫺6 mol/L to the medium and cells were then incubated in the presence of both drugs for a further 24 hours. We found that visfatin mRNA and protein were lower in the AG490⫹Ang II group, SP600125⫹Ang II group and U0126⫹Ang II group than Ang II group but higher than the control group. The AG490⫹Ang II group was lower than the SP600125⫹Ang II group and U0126⫹Ang II group at the
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mRNA and protein level of visfatin, and there was no significant difference between the SP600125 group and the U0126 group (Figures 4C and 4D).
DISCUSSION The cultured cardiomyocytes provide many advantages for developmental, physiological and pharmacological studies of cardiac tissue, because they are allowed for the direct cell manipulation and control of environmental parameters without interference from the compensatory feedback mechanisms that exist in vivo. The cardiomyocytes in the embryonic or neonatal rats are easy to be isolated and cultured, and their responses to external stimulus are more sensitive than the mature cardiomyocytes. Thus, most studies use these immature cells for studying pharmacology and intracellular signal transduction pathways. Volume 343, Number 3, March 2012
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In the study, in order to examine the visfatin expression in the cardiac cells, we isolated and cultured the cardiomyocytes and cardiac fibroblasts and examined their visfatin expression, respectively. We found that visfatin was expressed both in the cardiomyocytes and in the cardiac fibroblasts by means of RT-PCR and Western blot. Visfatin mRNA and protein expression in cardiac fibroblasts were expressed slightly more highly than in the cardiomyocytes, but there was no significant difference. BNP is widely distributed in the brain, spinal cord, heart and lung, among which the heart was the highest. Normally, the quantity of BNP is low in ventricle, but BNP gene expression is elevated rapidly during the wall tension rising. In this study, the cardiomyocyte hypertrophy was induced by Ang II, and we assessed the cardiomyocyte hypertrophy indirectly by examining the BNP protein expression in the cardiomyocytes. Visfatin and BNP expression were increased in a dose- and time-dependent manner in the cardiomyocytes pretreated with Ang II. The increased BNP protein expression suggested that cardiomyocytes had gradually transformed into hypertrophic cardiomyocytes. The increased visfatin expression may be related to the increased oxygen consumption of cardiomyocytes affected by Ang II. The visfatin gene is regulated by hypoxia inducing factor-1. There are two functional hypoxia response elements in the promoter region of the visfatin gene. The expression of visfatin increased through the interaction of hypoxia response elements and hypoxia inducing factor-1␣.24 After that, the increased visfatin expression may also be caused by the apoptosis of cardiomyocytes. The apoptosis of cardiomyocytes can be induced by Ang II,25 and it can be resisted by visfatin.26 Thereby, we deemed that the expression of visfatin was increased to resist the apoptosis of cardiomyocytes. Ang II induces cardiac remodeling through specific receptors, which act on the cardiac cells. Pharmacological approaches indicated the existence of two subtypes of angiotensin receptors, named Ang II types 1 and 2 receptors (AT1-R and AT2-R).27 AT1-R and AT2-R are both G-protein-coupled receptor family members,28 but recent studies show that roles of two kinds of receptors are different from each other.29 Telmisartan is the specific inhibitor of AT1-R, and PD123319 is the specific inhibitor of AT2-R. In the study, we found that overexpressed visfatin induced by Ang II was completely blocked by telmisartan, showing that visfatin was overexpressed through AT1-R but not through AT2-R. Ang II-induced cardiomyocyte hypertrophy was mainly mediated by the AT1-R.30 Positive chronotropic and inotropic effects of Ang II, which increased oxygen consumption, could be inhibited in the myocardium and vascular smooth muscle cells in which the AT2-R gene is overexpressed. Therefore, we speculated that the increased expression of visfatin induced by AT1-R mediated oxygen consumption in Ang II-induced cardiomyocyte hypertrophy. AT1-R can mediate the resisting of cardiomyocyte to the apoptosis and protein synthesis.31 On the contrary, AT2-R plays an important role in resisting the hypertrophy and promoting the apoptosis.32,33 The increased visfatin expression probably was mediated by AT1-R to resist the cardiomyoctye apoptosis in the cardiomyocytes pretreated with Ang II. The highest expression of visfatin in the PD123319⫹Ang II group blocked the effect of AT2-R on AT1-R, probably because of PD123319. Our data further confirmed the hypotheses that the elevated visfatin expression was induced by the increased oxygen consumption and apoptosis, which was Ang II induced. Ang II is a key effector molecule of the renin–angiotensin system (RAS) and acts to promote growth and proliferation through the activation of © 2012 Lippincott Williams & Wilkins
widespread signalling mechanisms. There is a substantial body of evidence indicating that this peptide contributes to changes in the cardiac structure and function. Abnormality of the RAS has been strongly implicated in the cardiomyoctye hypertrophy. The reasons for that AT1-R blocker reverses cardiomyocyte hypertrophy may partly because that it blocks the increased visfatin expression induced by RAS. AT1-R activates the specific tyrosine kinase pathway and causes tyrosine phosphorylation, and then ERK 1/2, JNK and JAK are activated. Studies showed that JNK could be activated by Ang II through AT1-R in the cultured neonatal rat cardiomyocytes.34 ERK is a key member of the mitogenactivated protein kinases family, which is closely related to Ang II-induced cardiomyocyte hypertrophy.35 The JAK/STAT pathway is another pathway closely related to the Ang IIinduced cardiomyocyte hypertrophy, which is different from the mitogen-activated protein kinases family. Recent studies have shown that the JAK/STAT signaling pathway plays a central role in the cardiac pathophysiology. In addition, JAK/ STAT signaling represents one limb of an autocrine loop for Ang II generation, which serves to amplify the action of Ang II on the cardiomyocytes.36 In this study, we found that the increased visfatin expression was blocked mainly by AG490, partly by U0126 and SP600125 in the Ang II-induced cardiomyocyte hypertrophy. It was probably because AG490 blocked the JAK/STAT-mediated autocrine loop for cardiac endogenous Ang II generation. All in all, these results suggested that visfatin expression was elevated mainly through JAK/STAT pathway in Ang II-induced cardiomyocyte hypertrophy. The OD values of MTT products of cardiomyocytes reached a maximum after stimulation by Ang II 10⫺6 mo1/L for 12 hours, and after that, as Ang II concentration and culturing time increased, the OD values significantly decreased, suggesting that the physiological function of cardiomyocytes was abnormal, and cardiomyocytes viability changed from compensation to decompensation. Christian et al20 found that plasma visfatin was significantly increased in the stable heart failure and increased further in the acute decompensation heart failure. At the cellular level, we also found that visfatin expression was higher when cardiomyocytes viability changed from compensatory to decompensation in the Ang II-induced cardiomyocyte hypertrophy. In conclusion, our data demonstrated that there was no significant difference of visfatin expression between cardiac fibroblasts and cardiomyocytes at the mRNA and protein levels. Visfatin expression was increased mainly through the JAK/STAT pathway in the process of the AT1-R-mediated cardiomyocyte hypertrophy, which was induced by Ang II. This study provided a novel area for future research into roles of visfatin in the process of cardiomyocyte hypertrophy. Further investigation would be needed to clarify the clinical significance of visfatin in the cardiomyocyte hypertrophy. ACKNOWLEDGMENTS The authors are grateful to all the staff of the Laboratory Animal Center, The Fourth Hospital of Hebei Medical University, for their excellent assistances in this study. REFERENCES 1. Ruzicka M, Leenen FHH. Relevance of angiotensin II for cardiac hypertrophy and failure induced by cardiac volume overload. Heart Fail Rev 1999;3:169 – 81. 2. Domenighetti AA, Wang Q, Egger M, et al. Angiotensin II-mediated
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Volume 343, Number 3, March 2012
Angiotensin II Type-1 Receptor-JAK/STAT Pathway Mediates the Induction of Visfatin in Angiotensin II-Induced Cardiomyocyte Hypertrophy Liang Chang, MM, Rong Yang, MM, Mei Wang, MM, Jinming Liu, MM, Yaling Wang, MM, Hui Zhang, MM and Yongjun Li, PhD
Abstract: Introduction: The new adipocytokine visfatin is closely associated with the cardiovascular diseases, and expression of visfatin is elevated in the heart failure patients. However, at the cellular level, little work has been done on visfatin expression in the cardiomyocyte hypertrophy. Here, the authors investigated the expression and mechanisms of visfatin in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy in vitro by means of the cultured neonatal rat cardiomyocytes. Methods: After primary culture of 2- to 3-day-old Sprague– Dawley rat cardiomyocytes and cardiac fibroblasts, cardiomyocytes were pretreated with Ang II. Ang II type-1 receptor (AT1-R) antagonist telmisartan and Ang II type-2 receptor antagonist PD123319 were used to block effects of Ang II. These inhibitors used for the AT1-R pathway determination included SP600125, AG490 and U0126. Cell viability was examined using the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The expression of visfatin was examined by means of reverse transcription-polymerase chain reaction and Western blot. The expression of brain natriuretic peptide was examined through western-blot analysis. Results: Visfatin was found expressed in cardiomyocytes as well as cardiac fibroblasts, and there was no significant difference at the mRNA and protein levels of visfatin. Ang II treatment induced the increased expression of visfatin and brain natriuretic peptide in a dose- and time-dependent manner in cardiomyocytes, and pretreatment with AT1-R antagonist telmisartan completely blocked Ang II-induced visfatin expression increasement. The increased visfatin expression was also blocked by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway inhibitor AG490. Conclusion: Visfatin expression was increased mainly through the AT1-R-JAK/STAT pathway in the process of Ang IIinduced cardiomyocyte hypertrophy. Key Indexing Terms: Visfatin; Myocyte; Hypertrophy; Angiotensin II. [Am J Med Sci 2012;343(3):220–226.]
T
he cardiomyocyte hypertrophy is one of the main ways in which the cardiomyocytes respond to the mechanical and neurohormonal stimuli. It is well known that sustained increasement against the systolic or diastolic wall stress will lead to the cardiomyocyte hypertrophy. Both in vitro and in vivo studies show that trophic responses to stretch (increases in diastolic wall stress) involve angiotensin II (Ang II).1 Ang II also exerts the direct growth-promoting effect on cardiac tissues, resulting in the cardiomyocyte hypertrophy and the mechanical dysfunction independent of pressure overload.2,3 Ang II activates several intracellular signal transduction pathways, and therefore the local increase in the Ang II concentration could significantly From the Department of Cardiology, The Second Hospital of Hebei Medical University; and The Hebei Institute of Cardiovascular and Cerebrovascular Diseases, Shijiazhuang, Hebei, China. Submitted March 3, 2011; accepted in revised form June 8, 2011. Correspondence: Yongjun Li, PhD, Shijiazhuang, Hebei 050000 (E-mail: [email protected]).
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contribute to the pathogenesis of cardiomyocyte hypertrophy, even in the absence of arterial hypertension.4 In addition, the cardiomyocyte hypertrophy is often accompanied by the increasement of protein synthesis and the overexpression of hypertrophic genes, such as brain natriuretic peptide (BNP).5,6 Normally, BNP is secreted mainly by the embryonic cells and adult ventricular myocytes. However, in the pathological cases, the ventricular myocytes transform from contraction type to synthesis type promoted by hypertrophy factors, and BNP gene expression increases. Hence, BNP has been recognized as the sign of cardiomyocyte hypertrophy.7 The adipose tissue is not only considered as the energy storage tissue but also a significant endocrine organ. Adipocytes secrete a series of vasoactive substances, named adipokines, such as leptin, tumor necrosis factor-␣, interleukin-6, resistin and adiponectin. Samal et al8 cloned a kind of protein from peripheral blood lymphocytes, which could promote the proliferation of pre-B cells, and therefore, they named it the pre-B-cell colony-enhancing factor (PBEF). Rongvaux et al9 demonstrated that PBEF was nicotinamide phosphoribosyltransferase, which was a cytosolic enzyme in nicotinamide adenine dinucleotide biosynthesis. Fukuhara et al10 reported a kind of adipocytokine, which was highly expressed in visceral fat, was called visfatin and corresponds to PBEF. However, recent studies contradict several previously reported results, showing that adipokines could be secreted and expressed not only in the adipose but also in the other organs. Meantime, tumor necrosis factor-␣, interleukins, adiponectin and leptin are synthesized and secreted not only in the adipose but also in the heart.11–15 The new adipokines visfatin can be expressed both in the visceral fat tissues and in the heart tissues.16 Visfatin is widely expressed in a variety of organs, which indicates its versatility. It is well known that visfatin plays an important role in the cardiovascular diseases.17 Visfatin is highly expressed in the lipid-rich macrophages and in patients with the acute coronary syndrome.18 Visfatin can promotes maturation of the vascular smooth muscle cells and contribute to the vascular remodeling.19 Plasma visfatin concentrations significantly increased in patients with heart failure.20 However, at the cellular level, little work has been done on visfatin expression in the cardiomyocyte hypertrophy. In addition, previous studies have found that visfatin was expressed in the heart, but so far, no any research focuses on expression of visfatin in the cardiac cells. Therefore, in this study, we examined expression of visfatin between cardiac fibroblasts and cardiomyocytes and investigated the expression of visfatin and its mechanisms in Ang II-induced cardiomyocyte hypertrophy in vitro by the cultured neonatal Sprague–Dawley rat cardiomyocytes.
The American Journal of the Medical Sciences • Volume 343, Number 3, March 2012
Visfatin Expression in Cardiac Hypertrophy
MATERIALS AND METHODS All experimental procedures in this study conformed to Hebei Province Experimental Animal Management Methods. No anesthetics were administered to avoid interferences with biochemical values. Preparation of Cardiomyocytes and Cardiac Fibroblasts and Cells Culture Ventricular cardiomyocytes were dissociated from the 2to 3-day-old neonatal Sprague–Dawley rats (The Laboratory Animal Center of Hebei Province, Shijiazhuang, China), preplated in 2 steps and plated onto 6 well microplates (Corning Costar, New York, NY) to yield confluent cardiomyocytes, as described previously.21 These cells were incubated in the culture medium with 10% fetal calf serum (Solarbio, Beijing, China) at 37°C in humidified air with 5% CO2. 5-bromo-2deoxyuridine (Santa Cruz Biotechnology, Santa Cruz, CA) 0.1 mM was routinely applied for 48 hours to inhibit cardiac fibroblasts growth.22 Forty-eight hours after seeding, the cardiomyocytes were cultured with the serum-free culture medium for another 24 hours, which were pretreated with Ang II (Sigma-Aldrich, St. Louis, MO). The neonatal rat cardiac fibroblasts were recovered from the preplating steps of the cardiomyocytes isolation and cultured in the maintenance media, which was the same as above. Upon proper confluence, the fibroblasts were split and passaged at a 1:2 ratio and used at passage one.23 Analysis of Cardiomyocytes Viability Effects of Ang II on cardiomyocytes viability were determined by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (HyClone, Logan City, UT). The cardiomyocytes were plated at a density of 1 ⫻ 105/mL in a 96-well plate. After 48 hours after seeding, the cardiomyocytes planted on 96-well plates (Corning Costar) were serum starved for another 24 hours before treatment with Ang II. Then, these cardiomyocytes were incubated with 20 L
of 5 mg/mL MTT solution for 4 hours at 37°C. Thereafter, 150 L of dimethyl sulphoxide (HyClone) was added to each well to dissolve the dye crystal formazan, and the plate was shaken for 10 minutes to make sure that all purple crystals were completely dissolved. The amount of MTT formazan was quantified by measuring the absorbance at 490 nm using a microplate reader. RNA Isolation and Reverse Transcription-polymerase Chain Reaction Analysis Total RNA was isolated from the cardiomyocytes and cardiac fibroblasts by TRIzol reagent (SBS, Beijing, China). The purity of RNA was assessed by measuring the absorbance at 260 nm. A specific 338-bp size fragment was amplified using the primers for examining visfatin gene expression (forward: 5⬘-ACTTTGAATGCCGTGAA-3⬘; reverse: 5⬘-AATCCAGTTGGTGAGCC-3⬘). Glyceraldehyde phosphate dehydrogenase expression was used as internal control (forward: 5⬘-GAGGCTCTCTTCCAGCCTTC-3⬘; reverse: 5⬘-AGGGTGTAAAAGCAGCTCA-3⬘), and the fragment of 380-bp size was amplified. The reverse transcription-polymerase chain reaction was run for 30 cycles under the following conditions. The DNA template was denaturated at 94°C for 60 seconds, specific annealing at 52°C for 60 seconds and 72°C for 60 seconds, with a final extension step at 72°C for 10 minutes. Amplification was linear under these conditions and was carried out in a Biometra T-gradient Thermoblock PCR System (Santa Cruz Biotechnology). All RT-PCRs for each gene were performed at the same time and with the same batch of Taq polymerase to reduce variations in RT-PCR efficiency. Band densities were quantified using a scanning densitometer coupled to scanning software Gel-Pro Analysizer 3.1 (Informax, Bethesda, MD). Western Blot Analysis The visfatin and BNP protein expression in the cardiomyocytes were examined using Western blot analysis. The cells were lysed with the radioimmunoprecipitation buffer (Sigma-
FIGURE 1. Cell morphology and visfatin expression in cardiac cells. (A) Cardiac fibroblasts 200⫻. (B) Cardiomyocytes 200⫻. Visfatin mRNA (C) and protein (D) expression in cardiac fibroblasts (1) and cardiomyocytes (2).
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FIGURE 2. Cell viability, visfatin and BNP expression in cardiomyocytes pretreated with different concentration of Ang II for 48 hours. (A) Analysis of cardiomyocytes viability with 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Visfatin mRNA (B) and protein (C) expression in cardiomyocytes: 1, control; 2, Ang II 10⫺8 mol/L; 3, Ang II 10⫺7 mol/L; 4, Ang II 10⫺6 mol/L; 5, Ang II 10⫺5 mol/L. (D) BNP protein expression in cardiomyocytes: 1, control; 2, Ang II 10⫺8 mol/L; 3, Ang II 10⫺7 mol/L; 4, Ang II 10⫺6 mol/L; 5, Ang II 10⫺5 mol/L. **P ⬍ 0.01; ANOVA for comparison against control group.
Aldrich). After centrifugation and quantification, the samples were boiled before use. For analysis, 80 g of each sample was loaded. Each sample were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto the polyvinylidene fluoride membrane (Millipore, Bedford, MA). The polyvinylidene fluoride membranes were then blocked by incubation with 5% skim milk (Becton, Dickinson and Company, Franklin, NJ) in 20 mM Tris 䡠 Cl, 150 mM NaCl, 0.05% Tween 20 tris-buffered solution (TBS) plus Tween 20 (TBS-T), pH 7.4, for 1 hour at room temperature. The membranes were then incubated with the primary antibody anti-PBEF (Santa Cruz Biotechnology; rabbit polyclonal 1:200 diluted in 5% TBS-T) and the primary antibody anti-BNP (Abcam, Cambridge, UK; rabbit polyclonal 1:500 diluted in 5% TBS-T) overnight at 4°C. These membranes were then washed thoroughly for 30 minutes at room temperature with TBST (0.1%) followed by incubation with the secondary antibody IRDye700DX and IRDye800CW (1:5000; Rockland, Gilbertsville, PA) (1:4000 dilution) for 1 hour at room temperature. The antibody complexes were visualized using chemiluminescence (Odyssey9120; LI-COR Corporation, Lincoln, NE). Band densities were measured using a scanning densitometer coupled to scanning software Gel-Pro Analysizer 3.1. These mem-
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branes were also reprobed with glyceraldehyde phosphate dehydrogenase (Santa Cruz Biotechnology) to determine the equal protein loading. Statistical Analysis Data were normalized for the housekeeping gene glyceraldehyde phosphate dehydrogenase and expressed as a mean ⫾ SD for 3 times experiments and analysis by t test and ANOVA, using SPSS 10.0 software. Differences were considered statistically significant when P ⬍ 0.05.
RESULTS Cell Morphology and Visfatin Expression in Cardiac Cells The cardiomyocytes (Figure lA) and cardiac fibroblasts (Figure lB) were observed under the inverted microscope (200⫻). As expected, PCR products amplified using the specific primers for visfatin showed a clear band at the predicted size of 338 bp both in the cardiomyocytes and cardiac fibroblasts (Figure 1C). Using Western blot analysis, visfatin protein expression was confirmed both in cardiomyocytes and cardiac fibroblasts (Figure 1D). As shown in Figure 1, however, there was no significant Volume 343, Number 3, March 2012
Visfatin Expression in Cardiac Hypertrophy
FIGURE 3. Cell viability, visfatin and BNP expression in cardiomyocytes pretreated with Ang II 10⫺6 mol/L for different times (6, 12, 24 and 48 hours). (A) Analysis of cardiomyocytes viability with 3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide. Visfatin mRNA (B) and protein (C) expression in cardiomyocytes: 1, 6 hours (a, control; b, Ang II); 2, 12 hours (a, control; b, Ang II); 3, 24 hours (a, control; b, Ang II); 4, 48 hours (a, control; b, Ang II). (D) BNP protein expression in cardiomyocytes: 1, control; 2, 6 hours; 3, 12 hours; 4, 24 hours; 5, 48 hours. **P ⬍ 0.01; ANOVA for comparison against control group.
difference in visfatin mRNA and protein expression between the cardiac fibroblasts and the cardiomyocytes. Ang II Induced an Increase in Cardiomyocytes Viability and the Expression of Visfatin and BNP The cardiomyocytes were treated with an increasing concentration of Ang II (10⫺8 mol/L, 10⫺7 mol/L, 10⫺6 mol/L and 10⫺5 mol/L) for 48 hours. The optical density (OD) values of MTT products of the cardiomyocytes pretreated with Ang II increased in a dose-dependent manner, peaking when the concentration of Ang II was 10⫺6 mo1/L, and it dropped significantly when the concentration of Ang II was 10⫺5 mo1/L (Figure 2A). At the mRNA level, visfatin expression was increased in a dose-dependent manner, which was the highest when the concentration of Ang II was 10⫺5 mo1/L, and it slightly lowered when the concentration of Ang II was 10⫺6 mo1/L, and there was no significant difference (Figure 2B). The same was for visfatin protein expression (Figure 2C). BNP protein expression increased in a dose-dependent manner, which was the highest when the concentration of Ang II was 10⫺6 mo1/L, and it significantly dropped when the concentration of Ang II was 10⫺5 mo1/L (Figure 2D). The cardiomyocytes were then treated with Ang II 10⫺6 mol/L at different times (6, 12, 24 and 48 hours). The OD values of MTT products of the cardiomyocytes were increased © 2012 Lippincott Williams & Wilkins
in a time-dependent manner, peaking at 12 hours, and it significantly decreased after 24 hours (Figure 3A). The cultured normal cardiomyocytes at different times (6, 12, 24 and 48 hours) were used as control groups. There was no difference at the mRNA and protein levels of visfatin at different periods between control groups. However, visfatin mRNA and protein expression were significantly higher than the corresponding control groups. Visfatin mRNA and protein expression were increased in a time-dependent manner, which were the highest at 24 hours, and significantly decreased after 48 hours (Figures 3B and 3C). BNP protein expression increased in a timedependent manner, which was the highest at 24 hours, and slightly decreased at 48 hours, but there was no significant difference between the 24-hour group and the 48-hour group (Figure 3D). Mechanisms of the Visfatin Expression Increasement in Ang II-Induced Cardiomyocyte Hypertrophy To investigate mechanisms of the visfatin expression increasement in the Ang II-treated cardiomyocytes, we blocked Ang II type-1 receptor (AT1-R) and Ang II type-2 receptor (AT2-R) activities by using telmisartan (10 mol/L) and PD123319 (10 mol/L), respectively. The cells were exposed to telmisartan and PD123319 30 minutes before the addition of AngII 10⫺6 mol/L to the medium and the cells were then
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FIGURE 4. Effects of Ang II receptor and AT1-R signalling inhibitors on visfatin expression. Visfatin mRNA (A) and protein (B) expression in cardiomyocytes treated with Ang II 10⫺6 mol/L for 24 hours in the presence of telmisartan (10 mol/L) or PD123319 (10 mol/L): 1, control; 2, Ang II; 3, telmisartan ⫹ Ang II; 4, PD123319 ⫹ Ang II. Visfatin protein (C) and mRNA (D) expression in cardiomyocytes treated with Ang II 10⫺6 mol/L for 24 hours in the presence of AG490 (20 mol/L), U0126 (20 mol/L) or SP600125 (20 mol/L). **P ⬍ 0.01; ANOVA for comparison against control group.
incubated in the presence of both drugs for a further 24 hours. We found that visfatin mRNA and protein expression were the highest in the PD123319 group, followed by the Ang II group, and at the same time, there was no significant difference between the telmisartan and the control groups (Figures 4A and 4B). These inhibitors used for the AT1-R pathway determination were SP600125 [c-Jun NH 2-terminal kinase (JNK)] (10 mol/L), U0126 [extracellular signal-regulated kinase (ERK1/ 2)] (10 mol/L) and AG490 [Janus kinase (JAK)] (10 mol/L). The cells were exposed to inhibitors 30 minutes before the addition of Ang II 10⫺6 mol/L to the medium and cells were then incubated in the presence of both drugs for a further 24 hours. We found that visfatin mRNA and protein were lower in the AG490⫹Ang II group, SP600125⫹Ang II group and U0126⫹Ang II group than Ang II group but higher than the control group. The AG490⫹Ang II group was lower than the SP600125⫹Ang II group and U0126⫹Ang II group at the
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mRNA and protein level of visfatin, and there was no significant difference between the SP600125 group and the U0126 group (Figures 4C and 4D).
DISCUSSION The cultured cardiomyocytes provide many advantages for developmental, physiological and pharmacological studies of cardiac tissue, because they are allowed for the direct cell manipulation and control of environmental parameters without interference from the compensatory feedback mechanisms that exist in vivo. The cardiomyocytes in the embryonic or neonatal rats are easy to be isolated and cultured, and their responses to external stimulus are more sensitive than the mature cardiomyocytes. Thus, most studies use these immature cells for studying pharmacology and intracellular signal transduction pathways. Volume 343, Number 3, March 2012
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In the study, in order to examine the visfatin expression in the cardiac cells, we isolated and cultured the cardiomyocytes and cardiac fibroblasts and examined their visfatin expression, respectively. We found that visfatin was expressed both in the cardiomyocytes and in the cardiac fibroblasts by means of RT-PCR and Western blot. Visfatin mRNA and protein expression in cardiac fibroblasts were expressed slightly more highly than in the cardiomyocytes, but there was no significant difference. BNP is widely distributed in the brain, spinal cord, heart and lung, among which the heart was the highest. Normally, the quantity of BNP is low in ventricle, but BNP gene expression is elevated rapidly during the wall tension rising. In this study, the cardiomyocyte hypertrophy was induced by Ang II, and we assessed the cardiomyocyte hypertrophy indirectly by examining the BNP protein expression in the cardiomyocytes. Visfatin and BNP expression were increased in a dose- and time-dependent manner in the cardiomyocytes pretreated with Ang II. The increased BNP protein expression suggested that cardiomyocytes had gradually transformed into hypertrophic cardiomyocytes. The increased visfatin expression may be related to the increased oxygen consumption of cardiomyocytes affected by Ang II. The visfatin gene is regulated by hypoxia inducing factor-1. There are two functional hypoxia response elements in the promoter region of the visfatin gene. The expression of visfatin increased through the interaction of hypoxia response elements and hypoxia inducing factor-1␣.24 After that, the increased visfatin expression may also be caused by the apoptosis of cardiomyocytes. The apoptosis of cardiomyocytes can be induced by Ang II,25 and it can be resisted by visfatin.26 Thereby, we deemed that the expression of visfatin was increased to resist the apoptosis of cardiomyocytes. Ang II induces cardiac remodeling through specific receptors, which act on the cardiac cells. Pharmacological approaches indicated the existence of two subtypes of angiotensin receptors, named Ang II types 1 and 2 receptors (AT1-R and AT2-R).27 AT1-R and AT2-R are both G-protein-coupled receptor family members,28 but recent studies show that roles of two kinds of receptors are different from each other.29 Telmisartan is the specific inhibitor of AT1-R, and PD123319 is the specific inhibitor of AT2-R. In the study, we found that overexpressed visfatin induced by Ang II was completely blocked by telmisartan, showing that visfatin was overexpressed through AT1-R but not through AT2-R. Ang II-induced cardiomyocyte hypertrophy was mainly mediated by the AT1-R.30 Positive chronotropic and inotropic effects of Ang II, which increased oxygen consumption, could be inhibited in the myocardium and vascular smooth muscle cells in which the AT2-R gene is overexpressed. Therefore, we speculated that the increased expression of visfatin induced by AT1-R mediated oxygen consumption in Ang II-induced cardiomyocyte hypertrophy. AT1-R can mediate the resisting of cardiomyocyte to the apoptosis and protein synthesis.31 On the contrary, AT2-R plays an important role in resisting the hypertrophy and promoting the apoptosis.32,33 The increased visfatin expression probably was mediated by AT1-R to resist the cardiomyoctye apoptosis in the cardiomyocytes pretreated with Ang II. The highest expression of visfatin in the PD123319⫹Ang II group blocked the effect of AT2-R on AT1-R, probably because of PD123319. Our data further confirmed the hypotheses that the elevated visfatin expression was induced by the increased oxygen consumption and apoptosis, which was Ang II induced. Ang II is a key effector molecule of the renin–angiotensin system (RAS) and acts to promote growth and proliferation through the activation of © 2012 Lippincott Williams & Wilkins
widespread signalling mechanisms. There is a substantial body of evidence indicating that this peptide contributes to changes in the cardiac structure and function. Abnormality of the RAS has been strongly implicated in the cardiomyoctye hypertrophy. The reasons for that AT1-R blocker reverses cardiomyocyte hypertrophy may partly because that it blocks the increased visfatin expression induced by RAS. AT1-R activates the specific tyrosine kinase pathway and causes tyrosine phosphorylation, and then ERK 1/2, JNK and JAK are activated. Studies showed that JNK could be activated by Ang II through AT1-R in the cultured neonatal rat cardiomyocytes.34 ERK is a key member of the mitogenactivated protein kinases family, which is closely related to Ang II-induced cardiomyocyte hypertrophy.35 The JAK/STAT pathway is another pathway closely related to the Ang IIinduced cardiomyocyte hypertrophy, which is different from the mitogen-activated protein kinases family. Recent studies have shown that the JAK/STAT signaling pathway plays a central role in the cardiac pathophysiology. In addition, JAK/ STAT signaling represents one limb of an autocrine loop for Ang II generation, which serves to amplify the action of Ang II on the cardiomyocytes.36 In this study, we found that the increased visfatin expression was blocked mainly by AG490, partly by U0126 and SP600125 in the Ang II-induced cardiomyocyte hypertrophy. It was probably because AG490 blocked the JAK/STAT-mediated autocrine loop for cardiac endogenous Ang II generation. All in all, these results suggested that visfatin expression was elevated mainly through JAK/STAT pathway in Ang II-induced cardiomyocyte hypertrophy. The OD values of MTT products of cardiomyocytes reached a maximum after stimulation by Ang II 10⫺6 mo1/L for 12 hours, and after that, as Ang II concentration and culturing time increased, the OD values significantly decreased, suggesting that the physiological function of cardiomyocytes was abnormal, and cardiomyocytes viability changed from compensation to decompensation. Christian et al20 found that plasma visfatin was significantly increased in the stable heart failure and increased further in the acute decompensation heart failure. At the cellular level, we also found that visfatin expression was higher when cardiomyocytes viability changed from compensatory to decompensation in the Ang II-induced cardiomyocyte hypertrophy. In conclusion, our data demonstrated that there was no significant difference of visfatin expression between cardiac fibroblasts and cardiomyocytes at the mRNA and protein levels. Visfatin expression was increased mainly through the JAK/STAT pathway in the process of the AT1-R-mediated cardiomyocyte hypertrophy, which was induced by Ang II. This study provided a novel area for future research into roles of visfatin in the process of cardiomyocyte hypertrophy. Further investigation would be needed to clarify the clinical significance of visfatin in the cardiomyocyte hypertrophy. ACKNOWLEDGMENTS The authors are grateful to all the staff of the Laboratory Animal Center, The Fourth Hospital of Hebei Medical University, for their excellent assistances in this study. REFERENCES 1. Ruzicka M, Leenen FHH. Relevance of angiotensin II for cardiac hypertrophy and failure induced by cardiac volume overload. Heart Fail Rev 1999;3:169 – 81. 2. Domenighetti AA, Wang Q, Egger M, et al. Angiotensin II-mediated
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