Li Sheng, Xu Yang, Ping Ye*, Yong-xue Liu, and Chun-guang Han

1Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China

2Department of Pharmacology & Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China

Effect of Atorvastatin on Expression of Peroxisome Proliferator-activated Receptor Beta/delta in AngiotensinⅡ-induced Hypertrophic Myocardial Cells In Vitro△

Li Sheng1?, Xu Yang1?, Ping Ye1*, Yong-xue Liu2, and Chun-guang Han2

1Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China

2Department of Pharmacology & Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China

peroxisome proliferator-activated receptor； cardiac hypertrophy； statin；angiotensinⅡ

Objective To explore the effect of atorvastatin on cardiac hypertrophy and to determine the potential mechanism involved.

Methods An in vitro cardiomyocyte hypertrophy from neonatal rats was induced with angiotensinⅡ（Ang Ⅱ） stimulation. Before AngⅡ stimulation， the cultured rat cardiac myocytes were pretreated with atorvastatin at different concentrations （0.1， 1， and 10 μmol/L）. The following parameters were evaluated: the myocyte surface area，3H-leucine incorporation into myocytes， mRNA expressions of atrial natriuretic peptide， brain natriuretic peptide， matrix metalloproteinase 9， matrix metalloproteinase 2， and interleukin-1β，mRNA and protein expressions of the δ/β peroxisome proliferator-activated receptor （PPAR） subtypes.

Results It was shown that atorvastatin could ameliorate Ang Ⅱ-induced neonatal cardiomyocyte hypertrophy in the area of cardiomyocytes，3H-leucine incorporation， and the expression of atrial natriuretic peptide and brain natriuretic peptide markedly. Meanwhile， atorvastatin also inhibited the augmented mRNA level of several cytokines in hypertrophic myocytes. Furthermore， the down-regulated expression of PPAR-δ/β at both the mRNA and protein levels in hypertrophic myocytes could be significantly reversed by atorvastatin treatment.

Conclusions Atorvastatin could improve AngⅡ-induced cardiac hypertrophy and inhibit the expression of cytokines. Such effect might be partly achieved through activation of the PPAR-δ/β pathway. Chin Med Sci J 2015； 30（4）:245-251

C ARDIAC hypertrophy is a fundamental response of cardiac myocytes to various stimuli， such as hypertension， ischemic heart disease， and valvular disease， and is believed to initially have a compensatory function which is associated with a significantly increased risk of heart failure and malignant arrhythmia.1，2In order to prevent cardiac hypertrophy， it is essential to identify the molecular events involved in the hypertrophic process. Although a plenty of signaling cascades have been implicated in the development of cardiac hypertrophy，3little is known about the intrinsic mechanisms with the potential to inhibit or even reverse hypertrophy.

It has been shown that inflammatory cytokines，including interleukin-1β，4matrix metalloproteinase （MMP）2， and MMP95are closely related to the occurrence and development of cardiac hypertrophy. As ligand-activated nuclear hormone receptors， the three isoforms （α， γ， and ?/δ） of peroxisome proliferator-activated receptors （PPARs）have recently been shown to play an important role in the control of inflammatory responses.6The α isoform is distributed in tissues with high rates of mitochondrial fatty acid-oxidation， the γ isoform is highly abundant in adipose tissue， and the ?/δ isoform is in ubiquitous tissues. Studies have indicated that activation of PPAR-α and PPAR-γ negatively regulates inflammatory signaling pathways， including nuclear factor-κB and activation protein-1， attenuates endothelin-1-induced hypertrophic gene expression， and increases cardiomyocyte size in vitro.7Unlike PPAR-α and PPAR-γ isoforms， the role of PPAR ?/δ in the heart is less clear. Recently， it has been shown that the selective PPAR-?/δ activator inhibits phenylephrine （PE）-induced hypertrophy in cultured neonatal rat cardiomyocytes.8

Statins， or 3-hydroxy-methylglutaryl-CoA （HMG-CoA）reductase inhibitors， are widely prescribed as cholesterollowering agents； statins competitively inhibit the activity of HMG-CoA reductase. Emerging data indicate that statins could attenuate cardiac hypertrophy in vivo.9，1OHowever，it is not clear whether the effect of statins on cardiac hypertrophy is related to PPARs or inflammatory cytokines and the underlying molecular mechanism is unknown. In this study， we examined the role of atorvastatin in suppression of cardiac hypertrophy in neonatal rat cardiomyocytes and investigated the potential mechanism involved.

MATERIALS AND METHODS

Materials

All cell culture reagents， including Dulbecco's Modified Eagle's medium （DMEM）， fetal calf serum （FBS）， and trypsin， were purchased from GIBCO （Burlington， Ontario，CA）. AngiotensinⅡ （Ang Ⅱ） was obtained from Sigma （St Louis， MO， USA） and 5'-bromodeoxyuridine （5'-BrdU） was purchased from the Aldrich Company （Gillingham， UK）. The RT-PCR kit and DNA Marker DL2OOO were obtained from Takara （Dalian， China） and3H-leucine from Beijing High Tech Co.， Ltd. （China）. Anti-PPAR-β/δ antibodies were from Santa Cruz Biotechnology （Santa Cruz， CA， USA）. Fluorescein isothiocyanate （FITC）-labeled secondary antibody was from Zhongshan Bio-tech Co.， Ltd. （Guangdong， China）. Atorvastatin was kindly provided by Pfizer Pharmaceuticals Limited （Dalian， China）.

Cell culture

Primary cultures of cardiac myocytes were prepared from the ventricles of Wistar rats （1-2 days of age） as previously described，11with minor modifications. Under germ-free conditions， minced ventricular myocardium was dispersed in O.O8% trypsin for approximately 5 minutes. This digestion step was repeated several times and the collected cell suspensions were mixed with culture medium （DMEM containing 1O% FBS）. The dissociated cells were collected by centrifugation at 157×g for 8 minutes at 4°C and resuspended in DMEM supplemented with 1O% FBS. The resuspensions were pre-plated for 1 hour in 5O ml culture bottles to remove any non-myocytes. The resultant cell suspension was plated in DMEM containing 5-BrdU （O.1 mmol/L） in a new culture bottle/dish for 48 hours at 37°C（5% CO2in air）. The culture medium was then changed to serum-free DMEM and the cells were pretreated without or with atorvastatin in different concentrations （O.1， 1， 1O μmol/L） for 3O minutes and subsequently stimulated with Ang Ⅱ （1 μmol/L） for 48 hours. The cultured cells without any treatment served as normal control.

Assay for cardiac myocytes surface areas

To assess the effect of atorvastatin on the size of myocytes，1OO cultured myocytes， which were randomly selected from 2 or 3 wells of every dish for each condition， were measured in terms of surface areas according to the method of Simpson et al12using Nikon camera （Nikon， JP）and analyzed with Tanon Gis-2OO8 （Shanghai， China）.

Incorporation of3H-leucine

As previously described，13cultured neonatal ventricular myocytes were plated at 5×1O5cells/ml （1.5 ml/well） in 12-well plates and treated with Ang Ⅱ （1 μmol/L） in the presence or absence of atorvastatin （1 μmol/L） and co-incubated with 37 kBq3H-leucine from culture days 2-4.At the end of the experiments， the cells were washed twice with phosphate buffered saline （PBS）， and then treated with 1O% trichloroacetic acid at 4°C for 1 hour to precipitate the protein. The precipitates were dissolved in NaOH（O.1 mol/L）. Aliquots were counted with a LS-65OO liquid scintillometer.

RNA preparation and RT-PCR analysis

Total RNA was isolated by TRIzol reagent （Invitrogen， CA. USA）. The extracted RNA was assessed by absorbance at 26O nm and 28O nm for quantity and purity with a DU-64O UV-spectrophotometer （Backman， CA. USA）.

The mRNA expressions of atrial natriuretic peptide（ANP）， brain natriuretic peptide （BNP）， interleukin-1β（IL-1β）， MMP2， MMP9， and PPAR-δ/β from cultured neonatal ventricular myocytes were determined by RT-PCR. The primers for amplification of the above genes are listed in Table 1. Primers were synthesized by Beijing SBS Genetech Co.， Ltd. （China）.

PCR was performed for 28-33 cycles， each cycle consisting of denaturation at 95°C for 45 seconds， annealing at 48°C-56°C for 1 minute， and extension at 72°C for 1 minute. The PCR product was separated using electrophoresis on 2% agarose gel and semi-quantified by an image analysis scanning system as a ratio to GAPDH （an internal reference）.

Immunofluorescence staining

Myocytes， which were plated on glass coverslips， were initially rinsed with DMEM for 3O seconds at ambient temperature and then fixed in 4% formaldehyde for 3O minutes， as previously described.14Cells were then washed three times in PBS before permeabilization and after each subsequent step. Permeabilization was performed in buffer consisting of 1.O% Triton for 3O minutes at room temperature. Coverslips were sequentially incubated with goat polyclonal antibody against PPAR-δ/β and FITC-labeled anti-goat antibody， each for 6O minutes at room temperature. Cells were then visualized and photographed using a Zeiss fluorescence microscope.

Statistical analysis

Statistical analysis was performed using SPSS statistical software. Data were presented as means ± standard deviation（SD） of triplicate experiments. Differences between various treatment groups were analyzed using one-way analysis of variance （ANOVA） and differences between two groups using Dunnett's t-test. P＜O.O5 was considered statistically significant.

Table 1. Primers of the objective genes

RESULTS

Effect of atorvastatin on hypertrophic myocytes

As shown in Fig. 1， the surface area of cardiomyocytes treated with Ang Ⅱ was significantly increased compared with normal ones， and atorvastatin pretreatment at a concentration of 1O μmol/L reversed the induction of Ang Ⅱcompared to dimethylsulfoxide （DMSO） （the solvent control）（P＜O.O1）. For3H-leucine incorporation， atorvastatin exhibited an inhibitory effect on hypertrophic myocytes （Fig. 2A）. The mRNA expression of ANP and BNP was markedly enhanced in Ang Ⅱ-induced myocytes compared with the normal controls， but atorvastatin down-regulated ANP and BNP mRNA in hypertrophic myocytes （all P＜O.O1， Fig. 2B）.

Effect of atorvastatin on the increased expression of cytokines in hypertrophic myocytes

MMP2， MMP9， and IL-1β mRNA expressions were increased in Ang Ⅱ-induced hypertrophic myocytes， while atorvastatin pretreatment resisted the increased expression （Fig. 3）

Effect of atorvastatin on expression of PPAR-δ/β in hypertrophic myocytes

The expression of PPAR-δ/β mRNA was reduced in hypertrophic myocytes， but was up-regulated with the administration of atorvastatin in a concentration-dependent manner （P＜O.O1， Fig. 4）. In a similar way， the AngⅡ-induced decrease in the expression of PPAR-δ/β protein was also improved by atorvastatin （P＜O.O1， Fig. 5）.

Figure 1. Effect of atorvastatin on the surface area of cardiomyocytes in vitro （n=1OO）.

Figure 2. Effects of atorvastatin on the incorporation of3H-leucine and mRNA expression of ANP and BNP in hypertrophic myocytes （n=3）.

DISCUSSION

Increasing evidence from clinical and experimental studies has shown that statins have pleiotropic effects beyond lipid-lowering. Nishikawa et al1Oreported that pravastatin or simvastatin is associated with a lower left ventricular mass in patients with angina. Similarly， Porter et al15showed that simvastatin reduces human atrial myofibroblast proliferation independent of cholesterol lowering in vitro. The mechanism by which statins prevent cardiac hypertrophy is not very clear. In this study， we focused on the effect of atorvastatin on cardiac hypertrophy in vitro and the potential molecular mechanism in cardiomyocytes.

Figure 3. Effect of atorvastatin on the expression of cytokines（n=3）.

Figure 4. Effect of atorvastatin on PPARβ/δ mRNA levels in cardiac myocytes in vitro （n=3）.

Cardiac hypertrophy is a final common way through which various cardiovascular diseases develop into heart failure. The characteristics of cardiac hypertrophy include not only the enlargement of cell size， but also the increase in constituent proteins， accompanied by complex changes in the re-expression of immature fetal cardiac genes， such as ANP and BNP. Inflammation is closely associated with the progressive pathologic development of cardiac hypertrophy and heart failure. It has been reported that IL-1β may play a role in ventricular remodeling with transition from left ventricular hypertrophy to congestive heart failure in the rat hypertensive model in vivo. Furthermore， in vitro，IL-1β induces unique cardiomyocyte hypertrophy and stimulates secretion of ANP and BNP from hypertrophic myocytes， the marker gene for cardiac hypertrophy.4MMPs are members of a large family of zinc-dependent enzymes， which specifically degrade extracellular matrix. During the course of left ventricular remodeling， the synthesis and secretion of MMPs in mature left ventricular myocytes accelerates.5The level of inflammatory cytokines in the heart is mediated through autocrine and paracrine mechanisms， which determines a direct role of cytokines in the development of cardiac hypertrophy. In the present study， Ang Ⅱ induced cardiomyocyte hypertrophy， characterized by an increased cell surface area and enhanced protein synthesis （i.e.，3H-leucine incorporation）， as well as an augmented expression of ANP and BNP. The expressions of IL-1β， MMP2， and MMP9 mRNA are also increased in hypertrophic cardiomyocytes， which is consistent with previous findings.16Atorvastatin treatment did reverse the above robust expression triggered by Ang Ⅱ in cardiomyocytes.

Figure 5. In vitro improvement of atorvastatin on PPAR protein expression in cardiac myocytes.

It is generally accepted that statin action against myocardial hypertrophy is attributed to the effect on small GTP-binding protein. However， most of the molecular mechanismshave not been delineated. In a β-myosin heavy chain-Q（4O3） transgenic rabbit model， simvastatin induced the regression of hypertrophy and fibrosis without an influence on the levels of Ras， Rac， and RhoA proteins， all being members of the small GTP-binding protein family.17These findings indicate that other signaling pathways， in addition to the small GTP-binding protein pathway， may be involved in the inhibitory effect of statins on myocardial hypertrophy.

PPARs are ligand-activated nuclear receptors， consisting of α， γ， and δ （or β） isoforms， having differential patterns of expression in the heart. It is well-established that PPARs play important roles in lipid and glucose metabolism. In addition， PPARs may regulate cell proliferation/differentiation and inflammation. It has been shown that PPAR-α and PPAR-γ activators inhibit cardiac expression of cytokines induced by lipopolysaccharide （LPS）.18It has been recently reported that a PPAR-δ-selective ligand inhibited LPS-induced tumor necrosis factor α （TNF-α）production from cardiomyocytes.18The inhibitory effects of the three PPAR isoforms activators on cardiac hypertrophy have been validated by others.19，2OThe similar effect between statins and PPARs on myocardial hypertrophy and inflammation suggests that there statins might have influence on PPARs. In rat hepatocytes， statin treatment results in a rise in PPAR-α mRNA levels both in vitro and in vivo and activates the mouse PPAR-α promoter in a reporter assay.21These findings demonstrate that PPAR-α might be a target gene of statins. It has been shown that pravastatin increased activity of PPAR-γ and suppressed nuclear factor-κB expression in monocytes.22In cultured human umbilical vein endothelial cells， statins significantly reduce interleukin-1β and -6 mRNA expressions and protein levels in the culture medium， while increasing PPAR-α and PPAR-γ mRNA and protein expressions.23Rosuvastatin increases vascular endothelial PPAR-γ expression in obese dyslipidaemic mice.24In the present study，atorvastatin diminished cardiac myocyte hypertrophy induced by Ang Ⅱ in a concentration-dependent manner，accompanied with up-regulation of PPAR-β/δ mRNA and protein expressions and down-regulation of levels of inflammatory cytokines in hypertrophied myocytes in vitro. Thus， atorvastatin might inhibit myocyte hypertrophy partly through a PPAR-β/δ signaling pathway， and then subsequently control the inflammatory response.

In conclusion， this study demonstrated that atorvastatin is effective in preventing the Ang Ⅱ-induced cardiac hypertrophy，during which PPAR-δ/β expression is up-regulated and the inflammatory cytokine response is attenuated in vitro. These results suggest a novel pharmacologic approach for the prevention and treatment of cardiovascular diseases characterized by cardiac hypertrophy. However， the information available about the signaling pathways linking statins，PPARs， inflammation， and cardiac hypertrophy is still lacking and further investigation is necessary.

REFERENCES

1. Levy D， Garrison RJ， Savage DD， et al. Prognostic implications of echocardiographically determined leftventricular mass in the Framingham-heart-study. N Engl J Med 199O； 322：1561-6.

2. Lorell BH， Carabello BA. Left ventricular hypertrophypathogenesis， detection， and prognosis. Circulation 2OOO；1O2：47O-9.

3. Hunter JJ， Chien KR. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 1999； 341：1276-83.

4. Harada E， Nakagawa O， Yoshimura M， et al. Effect of interleukin-1β on cardiac hypertrophy and production of natriuretic peptides in rat cardiocyte culture. J Mol Cell Cardiol 1999； 31：1997-2OO6.

5. Sakata Y， Yamamoto K， Mano T， et al. Activation of matrix metalloproteinases precedes left ventricular remodeling in hypertensive heart failure rats： its inhibition as a primary effect of angiotensin-converting enzyme inhibitor. Circulation 2OO4； 1O9：2143-9.

6. Staels B， Fruchart JC. Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes 2OO5；54：246O-7O.

7. Irukayama-Tomobe Y， Miyauchi T， Sakai S， et al. Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway. Circulation 2OO4； 1O9：9O4-1O..

8. Planavila A， Rodriguez-Calvo R， Jove M， et al. Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes. Cardiovasc Res 2OO5； 65：832-41.

9. Hayashidani S， Tsutsui H， Shiomi T， et al. Fluvastatin， a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor，attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation 2OO2；1O5：868-73.

1O. Nishikawa H， Miura S， Zhang B， et al. Statins induce the regression of left ventricular mass in patients with angina. Circ J 2OO4； 68：121-5.

11. Simpson P， McGrath A， Savion S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ Res 1982； 51：787-8O1.

12. Simpson P， Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyo-cardial cells. Circ Res 1982； 5O：1O1-16.

13. Thaik CM， Calderone A， Takahashi N， et al. Interleukin-1 beta modulates the growth and phenotype of neonatal rat cardiac myocytes. J Clin Invest 1995； 96：1O93-9.

14. Takemoto M， Node K， Nakagami H， et al. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest 2OO1； 1O8：1429-37.

15. Porter KE， Turner NA， O'Regan DJ. Simvastatin reduces human atrial myofibroblast proliferation independently of cholesterol lowering via inhibition of RhoA. Cardiovasc Res 2OO4； 61：745-55.

16. Frey N， Katus HA， Olson EN， et al. Hypertrophy of the heart： a new therapeutic target？ Circulation 2OO4；1O9：158O-9.

17. Patel R， Nagueh SF， Tsybouleva N， et al. Simvastatin induces regression of cardiac hypertrophy and fibrosis and improves cardiac function in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation 2OO1；1O4：317-24.

18. Ye P， Fang H， Zhou X， et al. Effect of peroxisome proliferator-activated receptor activators on tumor necrosis factor-alpha expression in neonatal rat cardiac myocytes. Chin Med J Sci 2OO4； 19：243-7.

19. Ding G， Cheng L， Qin Q， et al. PPAR delta modulates lipopolysaccharide-induced TNF alpha inflammation signaling in cultured cardiomyocytes. J Mol Cell Cardiol 2OO6；4O：821-8.

2O. Zhang H， Shao Z， Alibin CP， et al. Liganded peroxisome proliferator-activated receptors （PPARs） preserve nuclear histone deacetylase 5 levels in endothelin-treated Sprague-Dawley rat cardiac myocytes. PLoS One 2O14；9：e115258.

21. Paumelle R， Staels B. Cross-talk between statins and PPAR alpha in cardiovascular diseases： clinical evidence and basic mechanisms. Trends Cardiovasc Med 2OO8；18：73-8.

22. Lee TM， Chou TF， Tsai CH. Association of pravastatin and left ventricular mass in hypercholesterolemic patients：role of 8-iso-prostaglandin F2a formation. J Cardiovasc Pharmacol 2OO2； 4O：868-74.

23. Inoue I， Goto S， Mizotani K， et al. Lipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effect： reduction of MRNA levels for interleukin-1beta， interleukin-6， cyclooxygenase-2， and p22phox by regulation of peroxisome proliferator-activated receptor alpha （PPAR alpha） in primary endothelial cells. Life Sci 2OOO； 67：863-76.

24. Desjardins F， Sekkali B， Verreth W， et al. Rosuvastatin increases vascular endothelial PPAR-γ expression and corrects blood pressure variability in obese dyslipidaemic mice. Eur Heart J 2OO8； 29：128-37.

for publication April 7, 2015.

?These authors contributed equally to this article.

Tel: 86-10-88626575, Fax: 86-10-88270497, E-mail: yeping301@yahoo.com.cn

△Supported by the National Basic Research Program (973 Program) (2013CB530804).